FIELD OF THE INVENTION

The present invention provides a method for investigating neurovascular coupling. The present invention also provides in a separate embodiment, a method for monitoring and for evaluating the response to a treatment in a subject suffering from a cerebral small vessel disease.

BACKGROUND OF THE INVENTION

The cerebral microvasculature is responsible for delivering a continuous supply of energy to brain cells through two major, intricately regulated mechanisms (1): cerebral autoregulation, which ensures the maintenance of cerebral blood flow over a wide range of arterial pressure variations, and functional hyperemia, which ensures the rapid delivery of oxygen and glucose to active neurons. These mechanisms are engaged in response to complex signaling pathways elicited at the level of the neurovascular unit, comprising neuron terminals and astrocytes, as well as endothelial cells, smooth muscle cells and pericytes within the wall of microvessels (2). The biological processes underlying the changes in vascular diameter and resulting hyperemia (i.e. increase in cerebral blood flow) during neural activation are referred to as neurovascular coupling (NVC) (1).

Cerebral small vessel diseases encompass all pathological processes that affect small vessels of the brain, including small arteries and arterioles but also capillaries and small veins. Consequences of small vascular diseases (SVDs) in the brain parenchyma encompass various types of lesions including ischemic and hemorrhagic manifestations and diffuse white-matter changes. Chronic, diffuse and subclinical ischemia may lead to incomplete infarction (demyelination, loss of oligodendrocytes, axonal damage, etc.), while severe and localized ischemia may lead to focal complete necrosis in gray or white matter). Also vessel damage may lead to vessel rupture further leading to microscopic bleeding (microbleeds) or macroscopic hemorrhage with extensive tissue destruction (Pantoni L, Lancet Neurol 2010; 9:689-701).

SVDs are among the most prevalent neurological diseases and play a crucial role in at least three fields: stroke, dementia, and ageing. Indeed, Cerebral small vessel diseases (SVDs) are major contributors to stroke, disability, and cognitive decline that develop with aging (4). However, despite the enormous impact of SVDs on human health, the disease processes and key biological mechanisms underlying these disorders remain largely unknown. Accumulating experimental evidence suggests that functional or structural alterations in the cerebral microvasculature have early and deleterious consequences on brain tissue prior to or in association with the occurrence of focal ischemic or hemorrhagic lesions (5, 6). More attention and targeted efforts are therefore needed to better understand the pathogenesis of vascular injury to the brain caused by small vessel diseases in particular at their early stage. More particularly, specific preventive and therapeutic measures to reduce the burden of functional loss caused by small vessel disease remain to be designed. In this context, assessment of therapeutic trials in patients suffering from a cerebral small vessel disease remain complicated, the design of a method for monitoring the effects of a treatment in patient suffering from such disease is therefore of high relevance.

Indeed, while correlates between white matter lesions, lacunar infarct, microbleeds or large hematoma and small vessel diseases have been established using neuroimaging techniques such as for example MRI (magnetic resonance imaging), early surrogate biomarkers allowing the direct assessment of the microvasculature functioning that participate in the pathogenesis of brain damage are still required.

Neurovascular coupling is possibly impaired at early stages of cerebral small vessel or neurodegenerative diseases (Huneau et al., Front Neurosci 2015; 9:467). Changes in NVC in vivo can be probed using functional magnetic resonance imaging (fMRI) techniques (17). Most fMRI methods are based on changes in MR signals caused by hemodynamic variations at the cortical level that occur with a repeated neural task (18). Blood oxygenation level-dependent (BOLD) contrast, which is related to changes in blood volume and deoxyhemoglobin concentration, is the most frequently used fMRI method owing to its high signal-to-noise ratio. However, there are many hurdles for interpreting these previous fMRI data. For example, the use of BOLD contrast precludes determination of whether the reduced response is attributable solely to local changes in blood flow or to altered oxygen use or exchange (22). The BOLD response, which presumably originates from changes in blood volume in the venous compartment, also appears to be relatively insensitive to increases in capillary blood flow (23, 24). In addition, the possibility that a diminished neural response is the main driver of the reduced increase in flow observed in previous studies cannot be excluded in the absence of a separate, independent evaluation of neuronal activity. Finally, potential remote effects of deep ischemic lesions on cortical NVC, as well as the effects of multiple, currently used treatments, which can also alter the hemodynamic response, were not systematically excluded in these previous studies (25).

Therefore, an early surrogate biomarker based on cerebral blood flow variations remains to be identified. SUMMARY OF THE INVENTION

In response to a transient neural activity nearby vessels dilate, substantially increasing cerebral blood flow (CBF). This increase of CBF in response to transient neuronal activity is also referred to functional hyperemia. Surprisingly, the inventors have shown that the kinetics of the blood flow variation was altered in subjects suffering from a cerebral small vascular disease (CSVD).

They have used functional magnetic resonance imaging (fMRI) combined with cortical stimulations of various durations and have demonstrated that although baseline flow values did not significantly differ between patients and controls subjects, a significant change in the amplitude of hyperemia was detected in patients during the time course of stimulation, especially with longer stimulations as compared to shorter stimulations. The inventors therefore hypothesized that the dynamics, rather than the amplitude, of cerebral blood flow (CBF) increases (i.e.: functional hyperemia), in response to motor or visual stimulation, was particularly altered in patients with CSVD. The dynamics of the blood flow response during visual or motor stimulation was defined as the slope of functional hyperemia over at least one selected time segment.

In contrast no difference was observed in the same cortical regions when measuring evoked potentials during simultaneous and identical stimuli.

The results of the inventors provide evidence that the slope of the functional hyperemic response can be used as a biomarker of NVC alterations at early stages of CSVD, in particular at early stages of CADASIL.

Thus the invention relates to a method for investigating cerebral blood flow (CBF) in a subject comprising the steps consisting of:

(a) subjecting the subject to at least one stimulation, said at least one stimulation inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the CBF variation associated with the neural activation of said cortical region.

Typically, the kinetics of the CBF variation (also defined as the kinetics of the functional hyperemic response) can be defined as the slope of the CBF variation during the stimulation over at least one selected time window. More specifically the invention relates to a method for investigating cerebral blood flow (CBF) in a subject comprising the steps consisting of:

(a) subjecting the subject to stimulations of varying durations, said stimulations inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the CBF variation associated with the neural activation of said cortical region.

The invention also relates to a method for investigating neurovascular coupling in a subject during neural activation comprising the steps consisting of:

(a) subjecting the subject to at least one stimulation, said at least one stimulation inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the CBF variation associated with the neural activation of said cortical region.

In such a method the kinetics of cerebral blood flow variation is typically indicative of the quality of neurovascular coupling.

More specifically the invention relates to a method for investigating neurovascular coupling in a subject during neural activation comprising the steps consisting of:

(a) subjecting the subject to stimulations of varying durations inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the cerebral blood flow (CBF) variation associated with the neural activation of said cortical region,

In such a method the kinetics of cerebral blood flow variation is typically indicative of the quality of neurovascular coupling.

The invention further relates to a method for monitoring the effect of a treatment in a subject based on the kinetics of cerebral blood flow variation, wherein any one of the methods mentioned above is carried out at least once when the subject is receiving a treatment (i.e.: in a treated subject). DETAILLED DESCRIPTION OF THE INVENTION

Definitions according to the invention

The functional hyperemia corresponds to the variation of the cerebral blood flow (CBF) induced by a neural stimulation Neurovascular coupling (NVC) can be defined as the phenomenon that links a transient neural activity to the corresponding increase of CBF.

1. Methods for investigating cerebral blood flow and neurovascular coupling in a subject

In one aspect, the invention relates to a method for investigating the cerebral blood flow (CBF) in at least one subject comprising the steps consisting of:

(a) subjecting the subject to at least one stimulation period, said at least one stimulation period inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the CBF variation associated with the neural activation of said cortical region.

Typically, the kinetics of the CBF variation (also defined as the kinetics of the functional hyperemic response) can be defined as the slope of the CBF variation over at least one selected time window of the stimulation period.

In one embodiment of the method of the invention, at step a) the subject is subjected to two or more stimulation periods of varying durations inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition.

The invention also relates to a method for investigating neurovascular coupling in at least one subject during neural activation comprising the steps consisting of:

(a) subjecting the subject to at least one stimulation period, said at least one stimulation period inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the cerebral blood flow variation associated with the neural activation of said cortical region over variable durations,

Typically, the kinetics of the cerebral blood flow variation is indicative of the neurovascular coupling. Typically also, the kinetics of the CBF variation (also defined as the kinetics of the functional hyperemic response) can be defined as the slope of the CBF variation during the stimulation period over at least one selected time window.

In one embodiment of the method of the invention, at step a) the subject is subjected to two or more stimulation periods of varying durations inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition.

Typically in a method of the invention, the subject is subjected to at least one stimulating sequence inducing cerebral activation. The stimulation sequence comprises at least one, and preferably 2 or more stimulation periods. The stimulation periods can be of fixed or varying durations. Typically the stimulation periods are interleaved with resting periods. For example, the stimulation sequence may comprise alternating stimulating periods of varying durations, notably from 10 seconds to 80 seconds, preferentially from 10 to 60 seconds and more preferentially from 20 to 60 seconds or from 20 to 40 seconds. For example, said stimulations periods of various durations can therefore been selected from stimulations of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 seconds. The duration of the stimulation periods may be adapted typically as a function of the subject, the cortical area, the disease, and/or the state of the disease. At least one shorter and one longer stimulation periods are preferentially selected. For example the longer stimulation may last at least 30s, 35s, or 40s. Said stimulating periods of various durations are also preferentially randomly distributed.

Typically in a stimulating sequence, the at least one stimulation period, and typically the two or more stimulation periods of varying durations, is/are carried out alternatively with resting periods. For example a stimulating sequence may comprise randomly distributed stimulating periods of 20 and 40 seconds preferentially interleaved with resting periods. In another example the stimulation sequence can comprise one or more stimulation periods of a fixed duration as mentioned above. Typically in this embodiment, the duration of the stimulation period is of more than 20 seconds, preferentially more than 30 seconds, typically of 40 seconds. Preferentially the stimulation sequence comprises two or more stimulation periods interleaved with a resting periods. The resting period may typically last about 40 seconds. The stimulation sequence may be repeated 2 to 10 times.

A stimulation (i.e.: a stimulation period) according to the invention induces the activation of at least a cerebral region, typically a cortical region and preferentially a primary cortical region such as for example the primary motor cortex (Ml) and/or the visual (VI) area.

The activated cortical region is typically depending on the type of stimulation. Usually, the stimulation is a sensory and/or a motor stimulation such as for example a visual stimulation or a visually-cued motor task. For example the subject can be told to look at a screen, and to execute a specific motor task (such as a hand move) when a specific signal is displayed on the screen, while keeping rest when another signal is displayed.

According to the invention, the cerebral blood flow can be monitored using fMRI data acquisition based on arterial spin labelling (ASL), blood-oxygen level dependent (BOLD) or cerebral blood volume (CBV) MRI data. fMRI data acquisition is preferentially based on a method which allows a direct assessment of purely hemodynamics phenomena such as the cerebral blood flow such as arterial spin labelling (ASL). According to the invention ASL is selected from continuous ASL (CASL), pulsed ASL (PASL), pseudo-continuous ASL (PCASL) and velocity selective ASL (VSASL), most preferentially ASL is CASL.

Functional magnetic resonance imaging (fMRI) data acquisition of the activation cerebral region is performed while the subject is subjected to the stimulation as described above. For example, the field of view is typically positioned using the Ti volume for each subject to contain both the primary motor cortex (Ml) and visual (VI) area, when the subject is subjected to a visually-cued motor task.

The present invention is based on the measurement, through fMRI acquisition, of the kinetics of the variation of the CBF (which can also be named functional hyperemia as mentioned previously) induced by a stimulation period. As mentioned above successive stimulation periods of various durations are preferentially applied, but a stimulation period of a sufficient duration can be used. Indeed in this embodiment, the stimulation sequence may comprise one or more stimulation periods of a fixed duration. The one or more stimulation periods are typically interleaved with a resting period. A sufficient duration can be assessed as a duration allowing identification of a difference in the kinetics of the functional hyperemia induced by a stimulation of said duration, between a control healthy subject and a control subject suffering from a CSVD.

Step (b) of the methods of the invention then comprises the processing of imaging data acquired at step (a) in order to determine the kinetics of the CBF variation (i.e.: the signal variation when a technique allowing direct assessment of the CBF is used), associated with the neural activation induced by the stimulation period. Said kinetics is typically expressed as the amplitude of the CBF variation (signal variation) over a stimulation period. These values are preferentially recorded such that the method allows collecting information regarding the cerebral blood flow and the neurovascular coupling for a subject.

The kinetics of the signal's variation can be advantageously described using a linear mixed- effect model, as typically exemplified in the results. Said model may be applied over a selected time window during the stimulation period, notably a time window wherein a particular change in the kinetics of the CBF variation is observed (as illustrated in the examples). This selected time window may be identified visually. The one skilled in the art will understand that the exemplified model can be easily adapted to stimulation periods of various durations. Furthermore, the one skilled in the art will also understand that any appropriate model allowing to quantitatively describe the dynamics of blood flow variation can also be used.

The kinetics of the CBF variation (i.e.: the functional hyperemic response) can also be defined as the rope of the linear regression of relative CBF measured on a selected time segment of a stimulation period of a sufficient duration as defined previously. Typically said time segment is selected at least in the second third of the stimulation period. Preferably the stimulation period is comprised between 30 and 50 seconds, notably between 30 and 45 seconds, typically between 30 and 40 seconds. Typically a stimulation period of sufficient duration is of 40 seconds. The selected time-segment may last at least 10 seconds preferably 15 seconds.

It is to be noted that according to the invention the term "dynamics" may be used alternatively with the term "kinetics" as a synonym. At each time point of the stimulation period (preferentially at the end, or in the second half of the stimulation period), the ratio, or the difference, of both the amplitude of the reference CBF variation and the amplitude of the CBF variation for the subject may be determined and recorded for the matter of further comparison. Preferentially the stimulation period has a sufficient duration and has been shown to induce a different kinetics of the CBF variation as compared to the reference kinetics of the CBF variation obtained from at least a control subject as mentioned above.

Furthermore, in the methods according to the invention, the brain electrical activity can also be monitored simultaneously to fMRI data acquisition by measuring evoked potentials in stimulated cortical areas. Such simultaneous monitoring allows to control that, presumably, the CBF variations observed are not related to a reduction of neural activity occurring during stimulation.

Measurement of evoked potentials (i.e.: potential evoked by the stimulation, such as visual evoked potential when a visual stimulation is used) is typically obtained through an electroencephalogram (EEG) performed during fMRI acquisition. Typically, no difference in the same cortical region is expected to be detected, when measuring evoked potentials during simultaneous and identical stimulation periods.

Typically, the subject according to the invention is a mammal, such as a rodent, a feline, a canine, a bovine, an equine, a sheep, a porcine or a primate. Preferentially, the subject according to the invention is a human

According to the invention, the subject may be suffering from a cerebral small vessel disease or may be at risk of developing a cerebral small vessel disease (i.e.: is a presymptomatic individual).

Typically, the cerebral small vessel disease includes sporadic small vessel diseases related to age, hypertension or vascular risk factors; hereditary sporadic and hereditary cerebral amyloid angiopathy; inherited small vessel diseases distinct from cerebral amyloid angiopathy; inflammatory and immunologically mediated small vessel diseases, venous collagenosis or post radiation or toxic microangiopathy.

Small vessel diseases related to age, hypertension or vascular risk factors comprise notably fibrinoid necrosis, lipohyalinosis, microatheroma, microneursysms and segmental arterial disorganization.

Inherited small vessel diseases distinct from cerebral amyloid angiopathy comprise particularly CADASIL (cerebral autosomal dominant arteriopathy with subcortical ischemic strokes and leukoencephalopathy) CARASIL (cerebral autosomal recessive arteriopathy with subcortical ischemic strokes and leukoencephalopathy), hereditary multi-infarct dementia of the Swedish type, MELAS (mitochondrial encephalopathhy with subcortical ischemic strokes and leukoencephalopathy), Fabry's disease, hereditary cerebroretinal vasculopathy, hereditary endotheliopathy with retinopathy, nephropathy and stroke, and small vessel diseases caused by COL4A1 mutations. Inflammatory and immunologically mediated small vessel diseases comprise for example Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, Henoch- Schonlein purpura, cryoglobulinaemic vasculitis, cutaneous leukocytoclastic angiitis, primary angiitis of the CNS, Sneddon's syndrome, nervous system vasculitis secondary to infections, nervous system vasculitis associated with connective tissue disorders such as systemic lupus erythematosus, Sjogren's syndrome, rheumatoid vasculitis, scleroderma, and dermatomyositis.

Preferentially, a subject according to the invention is suffering from a cerebral small vessel disease related to hypertension or CADASIL. CADASIL is considered as a model of cerebral small vessel disease and mimics in particular the clinical and MRI manifestations observed in cerebral small vessel diseases related to hypertension.

The expression "at risk of of developing a CSVD refers to an individual who has not developed any of the cerebral features associated with a cerebral small vessel disease such as notably microbleeds, large hematoma, white matter lesions and/or lacunar infarcts.

The subject at risk of developing a CSVD can be a senior, or having hypertension, diabetes, atherosclerosis, insulin resistance, overweight, obesity, high triglyceride level, high fasting blood sugar or any further identified vascular risk factor increasing the risk of developing cerebral small vessel diseases. In one embodiment, the subject according to the invention suffers from a cerebral small vessel disease at an early stage. For example such a subject may exhibit loss or functional alterations of smooth muscle cells, lumen restriction, vessel wall thickening, vessel wall damage, microaneurysms and/or amyloid deposition.

The subject may be receiving a treatment, preferentially a candidate treatment, for a cerebral small vessel disease, or may have received a treatment for a cerebral small vessel disease. Therefore in some embodiment the subject is selected from a group a CSVD-treated subject.

Alternatively, the subject may be receiving a prophylactic treatment for a cerebral small vessel disease or may have received a prophylactic treatment for a cerebral small vessel disease. 2. Methods for monitoring the response to a treatment:

In a different embodiment, the invention also relates to a method for monitoring the effects of a treatment in a at least a subject, wherein the method as defined previously (notably the method for investigating the cerebral blood flow or neurovascular coupling) comprises the steps of

(a) subjecting the subject to at least one stimulation period, said at least one stimulation period inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the cerebral blood flow variation associated with the neural activation of said cortical region over variable durations,

Typically, the kinetics of the CBF variation (also defined as the kinetics of the functional hyperemic response) can be defined as the slope of the CBF variation during the stimulation over at least one selected time window. In one embodiment of the method of the invention, at step a) the subject is subjected to two or more stimulation periods of varying durations inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition.

Preferably the method is carried out at least once in a CSVD-treated subject (i.e.: when the at least one subject is receiving a treatment).

The said method can be carried out first in a non-treated subject (i.e.: in a subject that did not received any treatment, more specifically not treatment against CSVD, i.e. "non CSVD-treated" subject) and is repeated at least once in a CSVD-treated subject (i.e.: once the said subject is receiving a treatment or has received a treatment). Said method is therefore particularly useful for the assessment of therapeutic trials conducted on patients suffering from a cerebral small vessel disease. In this context the method is preferentially implemented on a group (or a population) of subjects.

As used herein, the expression "monitoring the response to treatment" has its general meaning in the art. It refers to the assessment of the evolution of a biomarker of cerebral small vascular diseases in a subject over time, in order to assess whether a given treatment has a beneficial effect or not.

Preferentially the said method is carried out at least twice during the time course of the treatment of the subject. The kinetics of the CBF variation can be recorded at each time for further comparison matter. Typically, the kinetics of the CBF variation is further compared at each implementation of the method with reference kinetics of the reference CBF variation. preferentially, the reference kinetics of a reference CBF variation may be obtained from at least one control subject; preferentially the reference kinetics is obtained from a population of control subjects typically as the averaged reference CBF variation (obtained from a population of control subjects) as a function of the stimulation time.

The reference kinetics may also be the kinetics of the CBF variation which has been obtained previously for the same subject or for the same group of subjects. Indeed, the reference kinetics may be the average CBF variation as a function of the stimulation time, which has previously obtained from the same population of subjects.

The kinetics of CBF variation and the reference kinetics are obtained during similar neural activations. In other terms, kinetics of CBF variation and the reference kinetics are obtained from fMRI data acquisition performed during a similar stimulation period and notably during a similar stimulation sequence.

The kinetics of the CBF variation obtained in a subject and/or used as a reference is advantageously described using a linear mixed-effect model as described previously, but any model allowing quantitative description of the dynamics of the CBF variation can also be used.

The use of such model allows quantitative testing of the difference of dynamics between the actual CBF variation and the reference CBF. The difference of kinetics as mentioned above (or the difference of CBF variation at selected time point of the stimulation sequence), established at each implementation of the method, is typically recorded and further compared for monitoring the effect of the treatment.

The kinetics of the CBF variation (i.e.: the functional hyperemic response) can also be defined as the rope of the linear regression of relative CBF measured on a selected time segment of a stimulation period or a sufficient duration as defined previously. Typically said time segment is selected at least in the second third of the stimulation period. Preferably the stimulation period is comprised between 30 and 50 seconds, notably between 30 and 45 seconds, typically between 30 and 40 seconds. Typically a stimulation period of sufficient duration is of 40 seconds.

Typically normalization, over the time course of the treatment, of the CBF kinetics obtained from the subject (or from a group of subjects) as compared to the reference kinetics of the CBF variation is indicative of a beneficial response to treatment. As used herein, the term "normalization" has its general meaning in the art. It refers to situations where a parameter or biomarker for a given defect (in the present situation the kinetics of the CBF variation in the patient) returns to a reference level (in the present situation to a reference kinetics for the CBF variation) observed in at least one healthy control subject or to a level closer to said control level. In other world, normalization can be understood as a decrease of the difference dynamics between the actual CBF variation and the reference CBF over the time course of the treatment.

In some cases an increase of the difference of dynamics may indicate that the treatment is deleterious.

It is also possible to compare over the time course of the treatment and typically at each implementation of the method as defined above, the amplitude of the CBF variation at a specific time point of the stimulation period, preferentially selected at the end of a stimulation period, with the reference amplitude of the CBF variation at the same time point of the same stimulation period. The reference amplitude of the CBF variation may have been previously obtained in the same subject (or group of subjects) or may be from at least one control subject.

Typically normalization over the time course of the treatment of the amplitude of the cerebral blood flow variation obtained from the subject as compared to the reference amplitude of the CBF variation is indicative of a beneficial response to treatment.

It is also possible to compare for the matter of monitoring of the effect of the treatment a ratio of both the amplitude of the reference CBF variation obtained from at least one control subject and the amplitude of the CBF variation for the at least one subject at the same specific time point of the same stimulation period established at each implementation of the method. The said ratio can be further compared, during the time course of the treatment and at each implementation of the method, with the ratio established at a prior implementation of the method.

Preferentially, said time point is selected at the end of the stimulation period.

Preferentially also, the stimulation period has a sufficient duration and has been previously shown to induce a difference in the kinetics of the CBF variation of at least one subject, as compared to a reference kinetics obtained from at least one control subject (notably before administration of the treatment).

As used herein the term "treatment" covers any type of treatment. It encompasses both prophylactic and curative treatments. The treatment can be an already known treatment used for cerebral small vessel disease or can be a candidate treatment, in the context of a therapeutic trial.

For example a treatment according to the invention encompasses treatments reducing risk factors for cerebral small vessel diseases (such as treatments selected from beta blockers, calcium channel blockers, antidiabetics, statins), drugs improving microcirculation (such as angiotensin-converting enzyme (ACE) inhibitors, antiplatelets or Angiotensin II receptor blockers (ARBs)) or that may be used in patients with cerebral small vessel diseases such as pain relievers, or antidepressants, as well as any other types of treatment envisaged for cerebral small vessel diseases as defined previously.

Advantageously, the method for monitoring the response to treatment according to the invention is non-invasive. It can be performed several times within a short interval on the same subjects without provoking any undesirable side-effects.

It is thought that the sensitivity of the method according to the invention is sufficient to detect even minor changes in dynamics of cerebral blood flow variations.

3. Biomarkers according to the invention:

The inventors have observed that, while there is no detectable change in the baseline blood flow values between patients suffering a from cerebral small vessel disease (especially at early stages) and control subjects, a significant change in the amplitude curve of hyperemia (herein a reduction) was observed in patients during the time course of stimulation periods of a sufficient duration, and in particular is observed during the time course of stimulation periods of longer duration as compared to shorter stimulation periods. Thus, in a separate embodiment, the present invention also relates to the use of the kinetics of cerebral blood flow variation in response to a cerebral stimulation, as a biomarker for cerebral small vessel disease, said kinetics of cerebral blood flow being determined through fMRI data acquisition on a subject subjected at least one stimulation period as defined above, wherein said stimulation period induces activation of a cortical region. In some embodiment, the subject is subjected to more than one stimulation period, typically two or more stimulation periods, of various durations as previously described.The definitions of the various parameters of this novel embodiment of the invention are the same as previously described.

4. Methods for detecting a neurovascular coupling impairment and for monitoring the course of the disease

In a different embodiment, the present invention also separately relates to a method for detecting a neurovascular coupling impairment in at least one subject. Said method is based on the method for investigating the neurovascular coupling as previously defined and comprises the steps of: (a) subjecting the subject to at least one stimulation period, said at least one stimulation period inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the cerebral blood flow (CBF) variation associated with the neural activation of said cortical region over variable durations;

the method further comprises a step of: (c) comparing said kinetics of cerebral blood flow variations associated with said neural activation of various durations with the reference kinetics of a reference blood flow obtained with similar neural activations;

wherein a difference between the at least one subject's kinetics of cerebral blood flow variation according to stimulation duration and the reference kinetics, during said neural activation is indicative of a neurovascular coupling impairment.

Preferably the at least one stimulation is of sufficient duration as defined previously.

Typically, the kinetics of the CBF variation (also defined as the kinetics of the functional hyperemic response) can be defined as the slope of the CBF variation during the stimulation over at least one selected time window.

In one embodiment of the method of the invention, at step a) the subject is subjected to two or more stimulation periods of varying durations inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition.

The present invention also separately relates to a method for detecting a cerebral small vessel disease a subject. Said method is also based on the method for investigating the neurovascular coupling as previously defined and comprises the steps of:

(a) subjecting the subject to at least one stimulation period of a sufficient duration, said at least one stimulation period inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition; and

(b) processing imaging data acquired at step (a) in order to determine the kinetics of the cerebral blood flow (CBF) variation associated with the neural activation of said cortical region over variable durations;

the method further comprises a step of:

(c) comparing said kinetics of cerebral blood flow variations associated with said neural activations of various durations with a reference kinetics of a reference blood flow obtained with similar neural activations;

Typically a difference between the at least one subject's kinetics of cerebral blood flow variation according to stimulation duration and the reference kinetics, during said neural activation indicates that the subject is suffering from a small cerebral vessel disease.

Typically, the kinetics of the CBF variation (also defined as the kinetics of the functional hyperemic response) can be defined as the slope of the CBF variation during the stimulation over at least one selected time window. In one embodiment of the method of the invention, at step a) the subject is subjected to two or more stimulation periods of varying durations inducing activation of a cortical region, while simultaneously performing functional magnetic resonance imaging (fMRI) data acquisition.

In one embodiment the present invention also relates to a method of treatment of a CSVD comprising the detection of a cerebral neurovascular coupling impairment or the detection of a CSVD as previously defined; and the treatment of said subject with at least one of the treatment as previously listed.

Parameters and definitions previously defined in the method for investigating neurovascular coupling in a subject can be applied to the present methods.

In particular, the kinetics of the CBF variation can be described as previously mentioned.

Typically the reference kinetics of a reference CBF variation may be obtained from at least one control subject; preferentially the reference kinetics is obtained from a population of control subjects typically as an averaged reference CBF variation (obtained from a population of control subjects) as a function of the stimulation time from a population of control subjects.

In this context the use of a model as mentioned above for quantitatively describing the dynamics of CBF variation allows to quantitatively test the difference of dynamics between a patient and a control subject or a population of control subjects.

The kinetics of CBF variation and the reference kinetics are obtained during similar neural activations. In other terms, said kinetics are obtained from fMRI data acquisition performed during a similar stimulation period.

In one embodiment, the method can be used for the monitoring of the course of the disease in at least one subject. Thus a method for monitoring a subject the method as mentioned above is repeated at least twice. Subject according to the invention have been described previously and are typically subjects which are suffering from a small vessel disease or which are at risk of suffering from a small vessel disease. The subjects may be receiving or not a treatment.

In the following, the invention will be illustrated by means of the following examples and figures. FIGURES:

Figure 1 : Neural stimulation procedures, acquisition protocol and selection of regions of interest.

(A) Visual and motor cortex areas were simultaneously activated with a visually cued motor task. The visual stimulation used was a black and white checkerboard flickering at 6 Hz. The motor task was an open-close hand movement performed at 1 Hz. (B) 20- and 40-second stimulation blocks were randomly distributed and interleaved with 40-second rest periods. (C) EEG recording during the fMRI experiment allowed measurement of the PI 00 wave (at 100 ms) from visual evoked potentials during visual stimulations. (D) Schematic summary of the pseudo-continuous arterial spin labeling (pCASL) fMRI imaging protocol and processing procedure. Labeling slice and imaged field of view of the pCASL sequence are shown projected on a Tl -weighted image from a control subject. ICA: independent component analysis.

Figure 2: Mean amplitude of functional hyperemia in visual and motor cortexes.

(A and B) ROIs projected on the surface of the primary visual cortex and motor (hand) cortex

(B) in a representative control subject. The ROI selected is shown in green, and calcarine and central sulci are marked by white dashed lines. (C and D) Average increase in CBF during 20- and 40-second stimulations in visual (C) and motor (D) ROIs are presented as violin plots for controls and patients, where points and lines represent means and standard deviations, respectively, and the width represents the frequency of data at different values. (E and F) Time series of functional hyperemia during 20- and 40-second stimulations in visual ROIs (E) and motor ROIs (F). Error bars represent standard deviations between subjects in each group. Dark gray bars represent the observed differences between mean values in control subjects and patients over each time frame.

Figure 3: Analysis of functional hyperemia dynamics and P100 waves during neural tasks.

(A and B) Functional hyperemia in the visual cortex (A) and sensorimotor cortex (B) during activation (after an initial 5-second period of rapid increase) was fitted using a piecewise (succession of 5-second steps) linear mixed-effects model in 19 patients and 19 controls. Likelihood ratio tests showed a significant difference in the dynamics (slopes) of the response between patients (red) and controls (blue) that was larger at the end phase of the stimulation period for long-lasting stimulations (**p < 0.01, ***p < 0.001). Changes in functional hyperemia dynamics were mainly detected between 15 and 30 seconds (yellow). Dark gray bars represent the difference between mean CBF values measured at different time intervals in control subjects and patients. (C) Average values (solid line) of evoked potentials (shown with their standard deviation, dotted line) obtained after each visual reversal stimulation did not differ between the two groups. (D) Analysis of PI 00 waves over 5-second segments using the piecewise linear mixed- effects model showed no significant difference over the entire duration of stimulation between the two groups.

Figure 4: Slope of functional hyperemia over the 15-30-second time frame in patients and controls.

An analysis of the CBF response, performed by comparing the slopes between 15 seconds and 30 seconds (yellow segment in figure 3) after the stimulus onset using a simple single-segment linear regression model, showed a significant difference between 19 patients and 19 controls in visual (p = 0.00723) and motor (p = 0.000907) ROIs. Similar results were obtained in an analysis of a replication sample of 10 patients and 10 additional healthy individuals. Conversely, results obtained using the same analysis of PI 00 amplitudes derived from EEG records in the visual cortex showed no significant difference between the two groups (p = 0.928).

EXAMPLE:

MATERIAL AND METHOD

Subjects:

CADASIL patients were included in the study based on the following criteria: 1) age between 30 and 60 years, 2) no current cognitive or motor complaints, 3) no significant disability and a modified Rankin Scale (mRS) of 0 or 1 , and 4) no focal neurological deficits at clinical examination. Age- and sex-matched healthy controls without any history of neurological disorder were recruited in parallel (see results paragraph). Neither patients nor controls had been treated with any antihypertensive agents or drugs with vasoactive properties (serotonin, dopamine, norepinephrine, phenylephrine or adrenaline) prior the MRI examination. The use of aspirin, clopidogrel, or antidepressant drug was tolerated. Current smokers could participate in the study, but only if they agreed to discontinue their tobacco use at least 1 day before the examination. Informed and written consent was obtained from all subjects. The study protocol was approved by an independent Medical Ethics Committee.

METHOD DETAILS

Stimulation protocol

The functional MRI study was based on repeated visually cued motor tasks (Figure 1A). Before the experiment, the neuroimaging examination and stimulation procedures were explained to each subject. Each participant was told to look at a screen that could be viewed via a mirror placed inside the MRI scanner. The subject was asked to perform simple opening- closing hand movements during the period when a flickering (6 Hz) black/white (100% contrast) checkerboard was displayed on the screen and to stop these movements as soon as a white cross was displayed on the black background. The subject had to use his/her non- dominant hand during all stimulation sequences, and was trained for a few minutes to perform the hand movements at a stable frequency of 1 Hz prior to commencing acquisitions. A total of six, 5-minute stimulation sequences were performed. Each sequence was composed of four activation periods lasting 20 seconds (n = 2) or 40 seconds (n = 2), randomly distributed and interleaved with four rest periods (Figure IB). The rest periods lasted 40 seconds plus a random jitter varying from 0 to 2.76 seconds (this upper limit corresponds to the repetition time of fMRI acquisition).

Electroencephalography

During ASL-fMRI experiments, electrical activity of the brain was recorded in all participants using electroencephalogram (EEG). Before the subject was placed inside the MRI scanner, they were equipped with an MRI-compatible, head-size-adjustable electrode cap (BrainCap-MR 64Ch-standard; EASYCAP Gmbh; Figure 1C). This cap allowed recording of 63 EEG (10-20 system) channels and contained a reference and a ground electrode positioned over the fronto-central cortex. It also had a separate electrocardiogram (ECG) channel with an external electrode that was placed under the left clavicle. During installation of the cap, electrical conductivity of each electrode at each location was checked using dedicated software. When needed, improvement was obtained by additional application of conductive gel (ECI Electro-Gel, MICROMED FRANCE SAS) at the corresponding location. Impedance between electrodes and the reference was kept below 10 kQ. Electrical signals were recorded at 5 kHz using two 32-channel amplifiers, with the battery placed close to the subject (BrainAmp MR plus; Brain Vision LLC) and linked to the BrainVision acquisition software. Electrophysiological recordings were performed during all fMRI sequences. MRI acquisition

Subjects were placed in a 3T MRI scanner (Magnetom Verio; Siemens Healthcare, Erlangen, Germany) and were first trained to perform hand movements while lying inside the MRI scanner. After subjects were positioned in the scanner, a full brain Tl -weighted magnetization-prepared rapid gradient echo (MPRAGE) was acquired using the following settings: repetition time (TR) = 2300 ms; echo time (TE) = 2.98 ms; slice thickness = 1 mm ³ ; 240 x 256 x 176 voxels. CBF and BOLD signal variations were simultaneously monitored using pseudo-continuous ASL (pCASL) with T2*-weighted echo planar imaging (EPI) (1), using the following settings: label duration = 1500 ms; post-labeling delay = 900 ms; TR = 2760 ms; TE = 10 ms. For each subject, the field of view (FOV; 6 slices, thickness = 7 mm, gap = 0.35 mm; 4 mm ² 64 x 64 matrix) was positioned using the Tl volume containing both the primary motor (Ml) and visual (VI) areas (Figure ID, left). In addition, two structural MRI sequences were acquired. Blood flow measurements were calibrated on a voxel-by-voxel basis using proton- density images (EPI; TR = 10000 ms; TE = 10 ms) obtained in the previous FOV T2*-weighted EPI images were obtained using the pCASL acquisition parameters (TR = 2760 ms; TE = 10 ms) over the entire brain (20 slices) to improve and facilitate the registration between fMRI volumes and the anatomic Tl volume.

QUANTIFICATION AND STATISTICAL ANALYSIS

MRI Data Analysis

Preprocessing

MRI preprocessing was performed using MATLAB with the SPM12 toolbox. All fMRI data were first realigned to correct for head movements that occurred during the study. The corresponding images were registered to Tl -weighted images (anatomy) using the full brain T2* sequence (see MRI acquisition, above). Calculation of blood flow signals was performed by subtracting the previous and subsequent tagged image from each control image, thereby preserving time sampling. CBF signals were then expressed as milliliters per gram per minute (ml/lOOg/min), assuming a blood-brain partition of 0.9 ml/g, a blood Tl of 1650 ms, and a labeling efficiency of 0.68 (26). Final CBF measurements were obtained by merging all fMRI data to yield a 654-volume time series. Regions of interest

An ROI was delineated in both VI and Ml areas based on the detection of blood flow changes that correlated with the stimulation paradigm (Figure ID, right). This detection was automated and was obtained separately for each subject using a spatial independent component analysis. First, merged blood flow series were bandpass filtered (cutoff frequencies: [0.01 ; 0.1] Hz) to eliminate remaining motion noise and slow variations. Filtered signals were then decomposed into 40 independent time-domain components based on the Infomax algorithm (44). The correlation of each component with the stimulation paradigm was then calculated. For the most-correlated components, the map of their distribution at the voxel level was used to determine activated voxels. The center of the FOV on the anteroposterior axis was used to separate the anterior and posterior parts of the stimulus-related map. The anterior part containing sensorimotor cortical areas was used to select voxels that were activated during the motor task, whereas the posterior part containing visual cortical areas was used to select voxels that were activated during the visual task. Each part was then normalized separately and used to build two z-score maps; this separation was performed based on the assumption that, with normalization, visual activation might alter the detection of motor activation, and vice versa. A Z-score threshold > 3 was chosen for delineating motor and visual ROIs in activated motor and visual areas in each subject. The detection of ROIs was performed using the MATLAB Software and the NEDICA toolbox (45). The sizes of ROIs in patient and control populations were compared using a two-sided Student's t-test. Finally, blood flow values for all voxels in each ROI were averaged to obtain the representative time series of CBF in motor and visual cortexes.

Statistical analysis of baseline blood flow

Resting state CBF during fMRI experiments was measured by averaging values obtained at all time points during baseline periods. These periods were defined as the time span from 30 to 40 seconds after the end of each stimulus. For each ROI, differences in resting CBF between groups were tested using the Wilcoxon non-parametric rank-sum test.

Modeling and analysis of functional hyperemia

For analysis of functional hyperemia (i.e., local blood flow responses to neural activation within motor or visual cortex areas), CBF data were first normalized to the mean resting CBF value obtained in each subject (percentage increase from baseline). Thereafter, the average CBF increase measured over the entire stimulation period (sum of all stimulation blocks) was compared between patients and controls using the Wilcoxon non-parametric rank- sum test.

For further analysis of blood flow responses, functional hyperemia dynamics was compared between patients and controls using a piecewise linear mixed-effects model, and CBF values were measured during the full activation periods; values obtained during the first 5 seconds of stimulation were discarded. This time-dependent model, which allowed investigation of changes in blood flow responses over different lengths of stimulation, is expressed as y„(t, fe, a) = m (t, k, a) + g(t, k, a). φ _η + U _n + R. _m , where

>'„ summarizes data variations in the n ^th subject as the sum of m(t, ft, a), a function modeling CBF changes during the different stimulation periods in each experiment, and g(t, k, a), a function modeling the difference in changes between patients and controls over the entire course of the experiment according to time (£) to stimulation sequence number k (relatively to the start of stimulation inside each stimulation block) and the age of the subject a (where a is between -15 and 15 and equals 0 for age = 45 years);

- m(t, k _r ) = β ₀ + k. p + α. β ₂ +∑! ^ [v^t in which β _α , β and β ₂ correspond to the initial CBF value, the hypothetical baseline blood flow variation along multiple repeated sequences and the effects of age on blood flow variations, respectively, and £ _έ represents the lower bounds of N in 5- second segments during the different stimulation periods (for 20-second blocks: N„ = 3 segments and = {5, 10,15} seconds; for 40-second blocks, N„ = 7 segments and t _t = {5,10,15,20,25,30,35} seconds; for the decrease between 15 and 35 seconds: N _p = 1 segment and t ₁ = 15), and where v _i models functional hyperemia dynamics in general;

- g(t, k, a) = i¾ + k. r + α. γ ₂ + Σ^&Ο - ¾} ⁺ Ι in which γ ₀ , γ ₁ and y ₂ corresponds to the initial CBF difference between patients and controls, the difference in CBF changes after repeated MRI sequences and the difference between groups of CBF variations related to the age effect, respectively, and where models the hypothetic difference in hyperemia dynamics between patients and controls;

φ _η is a binary independent variable equal to 1 for all patients and 0 for all controls;

U _n corresponds to the random effect for inter-subject baseline variability, where

R _n corresponds to the residual error of each measure, where R _n ^W ^" (0

The difference in the dynamics of CBF changes over time was used to test quantitative differences between controls and patients without a priori assumptions (except the choice of t _; ) using the maximum likelihood estimation method. Any subject with a Cook's distance value greater than 1 was assigned the first position (46), after which the likelihood ratio of the model, including those including one or several tested parameters versus a model that does not include these parameters, was calculated for testing different hypotheses. The analysis was performed using R Studio software with the 'influence.ME' package for computation of Cook's distance and the 'lme4' package for fitting linear mixed- effects models (47).

EEG analysis Preprocessing

Gradient/pulse and ballistocardiogram artifacts were first corrected using the average artifact suppression (AAS) method implemented in BrainVision Analyzer2 software (48), with application of an artifact waveform template of 5520 ms (2TR) averaged on a sliding window over 31 artifact repetitions. Signals were also filtered (low pass at 30 Hz with band rejection at 18 Hz) to suppress the remaining main and harmonic gradient frequencies. The signals were then down-sampled at 250 Hz. Ballistocardiogram artifacts were corrected using an artifact waveform template averaged on a sliding window over 21 cardiac cycles, assessed visually in the ECG channel. Eye-blink artifacts were automatically suppressed using an independent components analysis implemented in the Fieldtrip toolbox for MATLAB (49). Independent components were calculated from EEG sensors (Infomax algorithm) and correlated with the frontopolar EEG electrode with the lowest impedance between Fpl and Fp2. The component with the highest correlation was then suppressed when reconstructing EEG signals from all components.

Extraction of visual evoked potentials

The electrical visual response during the flickering checkerboard stimulation was evaluated based on the amplitude of the P100 wave extracted from the EEG signal obtained through the occipital EEG electrodes. P100 wave is a neural response that occurs around the calcarine fissure in response to a strong contrast change in the visual field (32-35). The signals of the three medial occipital channels, 01, Oz and 02, were averaged to obtain a single signal representative of primary visual cortex activity. For each scan of each subject, all responses evoked by individual checkerboard reversals were averaged to obtain the average latency of the PlOO wave. The PlOO amplitude was then calculated for each subject as the mean amplitude of each visual evoked potential (VEP) around the average latency (± 5 ms).

Modeling and analysis of PlOO amplitude

To evaluate possible alterations of the PlOO wave amplitude, we applied the piecewise linear mixed-model as used for blood flow analysis and described above, but where y _n (t, k, a) is the PlOO wave amplitude of the n ^th subject.

RESULTS

Study Design

A total of 19 CADASIL patients (11 females, 8 males) and 19 control subjects (12 females, 7 males) were initially included in the study. Mean age was 43.6 ± 6.7 years (range, 33-57 years) in patients and 43.2 ± 8.0 years (range, 30-58 years) in controls; all subjects but one, were right-handed. Rankin score at the time of MRI was 1 in eleven patients and 0 in the others; all control subjects had a Rankin score of 0. All patients presented with confluent white- matter hyperintensities on FLAIR (fluid attenuation inversion recovery) images, but showed no visually detectable regional or global cerebral atrophy on 3D-T1 images. Fifteen patients lacked lacunes, and four had from 1 to 3 lacunes. Microbleeds were absent except in one individual, who had two microbleeds. These signal abnormalities were absent in all healthy individuals.

Subjects performed a simple motor task with their non-directive hand that was visually cued by a flickering checkerboard (Figures 1A and IB) while undergoing an ASL sequence with simultaneous EEG recording. Activated voxels were detected individually in all subjects using a specific automatic method. Voxels that showed significant activation in response to visual stimulations were detected within the primary visual cortex, around and above the calcarine fissure, in all subjects. The size of visual regions of interest (ROI) was 70.5 ± 18.9 voxels (range, 36-100) in the patient group and 67.3 ± 25.1 voxels (range, 29-105) in the control group (p = 0.664, Student's test). Motor cortex activity was always detected in the contralateral hemisphere. In each case, the ROI observed around the central sulcus included a part of the primary motor cortex area and spread posteriorly to the central sulcus in the somatosensory area. The mean size of motor ROIs was 25.9 ± 9.1 voxels (range, 9-49) in patients and 28.6 ± 13.8 voxels (range, 3-60) in controls (p = 0.484). Comparison of Mean Variations in CBF between Patients and Controls Over 20- and 40- second Stimulations in Visual and Sensorimotor Cortexes

Resting CBF did not differ between patients and controls in the visual (56.5 ± 20.5 and 54 ± 13.5 ml/lOOg/min, respectively) or motor (52.8 ± 19.2 and 53.3 ± 15.8 ml/lOOg/min, respectively) cortex (p = 0.587). Changes in CBF in response to stimulations were calculated only in visual and motor ROIs. The mean increases in CBF over 20- and 40-second stimulations in visual and sensorimotor cortexes are shown in Figures 2C and 2D. Although there was a trend toward diminished functional hyperemia in patients compared with control subjects over 20-second stimulation blocks, these differences did not reach statistical significance (visual, p = 0.151 ; motor, p = 0.181). During 40-second stimulations, a small, but significant, reduction in the blood flow response was observed in motor ROIs (p = 0.027), but not in visual ROIs (p = 0.189).

Analysis of Functional Hyperemia Dynamics Over 20- and 40-second Stimulations in Visual and Sensorimotor Cortexes Using a Piecewise Linear Mixed-Effects Model

Despite the small difference in mean CBF change and large variability of CBF values measured during the experiment, we noted that the largest differences between patients and controls occurred predominately after 20 seconds in both the visual and motor cortex (Figures 2E and 2F, see bar plots of differences vs. time). Thus, we hypothesized that the dynamics, rather than the amplitude, of CBF increases in response to motor or visual stimulation was particularly altered in CADASIL. To analyze the dynamics of blood flow responses in patients and controls in a time-independent manner, we built a piecewise, linear mixed-effects model to fit functional hyperemia. In this model (detailed in STAR Methods online), the dynamics of the blood flow response was defined as the slopes over successive 5-second time segments (the limit of the time resolution of our recordings). Various potential non-dynamic effects on mean CBF measures due to age, the number of MRI sequences or inter-subject variability (modeled as a random effect) were thus considered with no other assumptions regarding the intrinsic dynamics of data apart from time sampling (Figure 3).

This analysis, which focused on changes detected during activation periods from the initial CBF rise (assumed to be complete 5 seconds after stimulation onset) to the end of neural stimulation, showed that functional hyperemia dynamics differed between patients and controls (Table 1). This difference was significant in both visual (p = 0.000736, likelihood-ratio test) and motor (p = 0.000001) ROIs over 40-second stimulation periods, but only in visual (p = 0.00278), and not motor (p = 0.125) ROIs with 20-second stimulations (Figures 3A and 3B and Table 1). Blood flow responses to repeated motor-stimulation sequences over 40 seconds also significantly decreased in patients (p = 0.0011), but no such decrease was detected for long (40 seconds) visual stimulations.

Table 1: Summary of group effects tested using a piecewise linear mixed-effects model in 19 patients and 19 age-matched controls (initial sample): CBF variations measured during 20- and 40-second activations of the visual and sensorimotor cortex were tested. PI 00 amplitude in the visual cortex in response to 20- and 40-second visual stimulations were also tested. Df, degree of freedom. Estimated values (Est.) are only provided for effects related to a single parameter (i.e., Df = 1). Red values indicate p-values less than 5% based on likelihood ratio tests. Comparison of the Slopes of Functional Hyperemic Responses in Sensorimotor and Visual Cortexes Over the 15-30-second Time Frame between Patients and Controls

An analysis of the time course of functional hyperemic responses during 40-second stimulations in both motor and visual ROIs showed a decrease in the CBF response in patients during the second half of the stimulation period (Figure 3A and 3B). This difference increased after 15 seconds in both visual and motor ROIs (Figures 3A and 3B, histograms). The decrease in the CBF response was analyzed by comparing the slopes of a simple, single-segment linear- regression model applied to CBF values between 15 and 30 seconds after the stimulus onset (Figures 3A and 3B, yellow time segment), considered as a single 15-second segment in Figure 4. Slopes calculated in patients and control groups were significantly different in both visual (p = 0.00723) and motor (p = 0.000907) ROIs.

Analysis of P100 Waves from Simultaneous Evoked Potentials Obtained with 20- and 40- second Visual Stimulations

The neural response evoked by visual stimulations was assessed in parallel with measures of evoked potentials and the amplitude of their derived PI 00 waves, which are known to originate from the calcarine fissure (Figures 3C and 3D). The average amplitude of P100 measured over 20 seconds after each visual contrast reversal did not differ between patients (1.7 ± 2.3 μν) and controls (1.9 ± 2.1 μν; p = 0.65; Wilcoxon signed-rank test); peak delay was also not significantly different between patients (0.108 ± 0.013 seconds) and controls (0.109 ± 0.0094 seconds; p = 0.73). Similarly, patients and controls showed no differences in average amplitude (2.52 ± 1.51 vs. 2.39 ± 1.22 μν; p = 0.81) or peak delay (0.110 ± 0.013 vs. 0.110 ± 0.009 s; p = 0.9) over 40 seconds (Figure 3C). Using the piecewise linear mixed- effects model based on successive 5-second time frames used previously for analyzing CBF values, we found no significant difference in the dynamics of PI 00 waves between patient and control groups for either 20-second (p = 0.329) or 40-second (p = 0.905) stimulations (Figure 3D). A comparison of the slopes of PI 00 responses derived from EEG records in the visual cortex over the 15-30-second time segment, as was previously done for CBF values in the simplified model, show no significant difference (p = 0.928) (Figure 4).

Analysis of the Slope of Functional Hyperemic Responses Limited to the 15-30-second Time Frame in a Replication Sample and Data Obtained in the Full Sample

We hypothesized that the slope of the functional hyperemic response could be a biomarker of NVC alterations at an early stage of CADASIL. To test the replicability of the slope difference between patients and controls over the 15-30-second time frame, we analyzed a separate sample of 10 CADASIL patients (mean age, 47.4 ± 3.5 years; range, 42-53 years; male/female ratio = 6/4) and 10 age-matched healthy subjects (mean age, 47.5 ± 5.1 years; range: 41-56 years, male/female ratio = 3/7) using selection criteria identical to those for the initial study population. A similar, but shorter, experimental paradigm composed of only four stimulation sequences was used for these analyses; data were subsequently processed as described for the more complex paradigm. Visual ROIs comprised 67.4 ± 19.7 voxels (range, 32-95) in patients and 77.7 ± 16.0 voxels (range, 62-102) in age-matched controls. With visual stimulations over 40 seconds, the slope of the functional hyperemic response between 15 and 30 seconds was lower in CADASIL patients (-24.4%) than in healthy subjects (-5.2%, p = 0.0238) (Figure 4 and Table 2). The size of motor ROIs was 17.6 ± 11.5 voxels (range, 6-37) in patients and 23.6 ± 15.4 voxels (range, 2-55) in controls, values that are smaller than those obtained in the initial sample. With a 40-second motor stimulation, the hyperemic response between 15 and 30 seconds in the sensorimotor cortex decreased in patients (-11.9%), but increased in controls (+ 8.9%, p = 0.0254) (Figure 4 and Table 2). In this additional sample, neural activity, measured using PI 00 amplitudes, did not differ between patients and controls (p = 0.944) (Figure 4). An analysis performed as previously, but using data from the entire sample of patients and controls (n = 58) showed that the slope of the linear regression based on CBF values measured between 15 and 30 s also differed between the two groups in both visual (p = 0.00000046) and motor (p = 0.0000012) ROIs (Table 2). Finally, the complete piecewise model was also fitted to the full dataset. This analysis confirmed a difference in functional hyperemia dynamics that remained highly significant in response to 40-second stimulations (visual, p = 1.28 x 10 ^"5 ; motor, p = 1.18 x 10 ^"6 ), but not to 20-second stimulations (visual, p = 0.133; motor, p = 0.431).

Table 2: Estimation and likelihood ratio test of the slope of the decrease in the functional hyperemic response between 15 and 30 seconds after stimulus onset: Results obtained in the initial sample (19 patients and age-matched controls) in a replication sample of 10 patients and age-matched controls, and in the whole sample (29 patients and 29 age-matched controls) are presented. Red values indicate p-values less than 5% based on likelihood ratio tests. (Est., estimated values).

DISCUSSION

In this study, we detected significant alterations in NVC at an early stage of CADASIL using ASL-fMRI in combination with EEG. These results were obtained in patients with a mean age of about 43 years, long before the occurrence of disability and cognitive decline that develop with cerebral atrophy at the latest stage of the disease (8). Alterations in functional hyperemia were invariably detected with both visual and motor stimulations, but only with longer (40 seconds) stimulations. A specific mathematical model showed major changes in functional hyperemia dynamics in patients, manifesting as a decrease in blood flow response after a delay of at least 15 seconds. With inclusion of additional data from a replication sample, the slope of blood flow curves between 15 and 30 seconds emerged as a potential biomarker of NVC dysfunction at an early stage of the disease.

An assessment of mean changes in absolute CBF values over 20 or 40 seconds of visual or motor stimulation revealed a significant decrease in functional hyperemia in 19 CADASIL patients compared with the same number of age-matched healthy individuals only for long- lasting motor tasks. Resting CBF values in these patients were in the normal range, as previously reported in the cortex at the onset of the disease (30). These results were obtained despite the large inter-subject variability of CBF measures over the cortex and the use of variable-sized ROIs, selected from stimulus-based maps. To assess hyperemia response dynamics, we developed a specific mathematical model that allowed analysis of functional hyperemic responses over successive 5-second time frames during stimulation periods while accounting for potential effects of age or the repetition of tasks throughout the duration of experiment. Applying this model, we found that the dynamics of blood flow responses differed substantially between patients and healthy individuals. This difference was highly significant for both repeated visual and motor tasks, but only for 40-sccond stimulations. Notably, this decrease in functional hyperemia was detected after a delay of 15 to 20 seconds in the patient group, both in the visual and sensorimotor cortex. Thus, the decline in hemodynamic responses across repetitive motor-stimulation sequences in the patient group revealed by the statistical model cannot account for the differences in hemodynamics. Moreover, this effect was not detected for long-lasting visual stimulations. Additional investigations are needed to determine whether these effects in patients might be explained by a greater fatigue factor associated with repeated hand movements over a long time compared with less demanding visual stimulations.

To assess neural activity using an independent approach, we obtained EEG recordings from patients inside the MRI scanner during each visual stimulation period. Our analysis of evoked potentials at the peak (observed at -100 milliseconds), which occurred after each visual contrast reversal (PI 00), revealed no significant differences between patients and healthy individuals with 20- or 40-second visual tasks, a result in line with a previous report based on three CADASIL cases (31). Both the average amplitude and delay to the peak were statistically indistinguishable between the two groups. Moreover, an analysis of changes in PI 00 over successive 5-second time frames using our piecewise linear mixed-effects model showed no significant change in PI 00 waves, regardless of the duration of visual stimulation. Since the PI 00 wave originates from the calcarine fissure, where the largest changes in blood flow are also detected during visual stimulations (32-35), our findings support the conclusion that the altered dynamics of blood flow responses during neural tasks in patients is not related to a reduction in the neural response, but may rather reflect a decrease in NVC efficiency originating from the vascular bed in the context of neural stimulations that extend over time.

On the basis of these findings, we hypothesized that the altered NVC dynamics observed in CADASIL patients might reflect early dysfunction of the cerebral microvasculature at the onset of the disease, and as such could be translated into a potential biomarker in future studies. A simplified analysis in which the linear slope of functional hyperemia was calculated over a single time frame from 15 to 30 seconds during stimulation periods rather than over 5- second steps not only confirmed these results in the original sample of 19 patients and 19 controls, but also in a replication sample of 10 additional patients and 10 age-matched controls. Moreover, a parallel analysis of PI 00 waves from visual evoked potentials over the same time frame showed no difference between the two groups in either the initial or replication samples. Finally, and as expected, in the whole population (29 patients and 29 controls), considering functional hyperemia over 15-30 seconds as a single marker confirmed a strong and highly significant difference between the two groups. While no marker of vascular dysfunction that affects primarily smooth muscle cells and pericytes has yet been identified in CADASIL, these data suggest that a measure as simple as the slope of the blood flow response obtained with fMRI over a single, delayed time frame represents a promising biomarker of early microvascular dysfunction in CADASIL. Additional investigations are now needed to determine whether and how this measure could be made sufficiently robust and sensitive to assess disease progression and clinical severity.

Studies have consistently reported a significant reduction in the amplitude of the CBF response in CADASIL mouse models. For example, CBF increases in the somatosensory cortex elicited by repeated whisker stimulations over 60 seconds were reported to be reduced by 30% in these mice (15). Notably, these results were obtained from mice that were 6 months old, simultaneously to the appearance of cerebral tissue lesions (14, 36). Accumulation of TIMP3 (tissue inhibitor of metalloproteinases-3), a component of the extracellular matrix in the wall of microvessels, was recently shown to disrupt signaling of the matrix metalloproteinase, AD AMI 7, resulting in an increase in the number of voltage-dependent potassium channels at the membrane surface of smooth muscle cells (14, 16). These early changes likely contribute to the reduced response to neural stimuli in CADASIL model mice (14). In the present study performed in CADASIL patients, all of whom were all at early stage of the disease but showed white-matter lesions on MRI, functional hyperemia dynamics were found to be altered at the late phase of long stimulations, whereas the amplitudes of responses were not different or only differed slightly between patients and healthy individuals. The contrast between these findings and preclinical results might be explained by large differences in experimental conditions, stimuli, time scales, signals produced, and underlying neurophysiological characteristics. We also cannot exclude the possibility that the much larger variability of CBF measures obtained using ASL-fMRI techniques in humans precluded the detection of modest differences in local CBF changes between patients and controls. Our innovative approach using a dedicated model for analyzing blood flow response-time curves in humans might have been particularly helpful in avoiding this pitfall by focusing on early changes in the shape of the response curve that are identifiable long before the decrease in amplitude becomes significant. Alternatively, it is conceivable that the reductions in blood flow responses in CADASIL patients detected with long stimulations are related to mechanisms that are specifically involved at a particular stage of the NVC process. Accumulating evidence indicates that NVC is initially composed of a fast, phasic component that reflects a hyperpolarizing wave initiated in capillary endothelial cells that rapidly back-propagates along the ascendant vascular tree to arteriolar smooth muscle cells (37). This process may also be accompanied by direct hyperpolarization of smooth muscle cells by activated astrocytes (38). By contrast, the mechanisms involved in sustaining the hemodynamic response during prolonged neural stimulation are not fully understood, although recent data suggest that a significant proportion of astrocytes that were not previously involved are progressively recruited into action by the release of glutamate from neurons within several seconds of the stimulation onset (39-41). Alterations in communication and signaling secondary to NOTCH3-ECD accumulation between astrocytic endfeet and vascular smooth muscle cells or pericytes (12) might thus be an additional contributor to the decreased efficiency of NVC with long neural stimulations.

This is the first report of precise CBF measures obtained in vivo using ASL, which showed that functional hyperemia dynamics are altered early in patients with ischemic cerebral SVDs. In a previous, small fMRI study of five CADASIL patients with a mean age of 55 years, the BOLD response stimulated in cortical areas was found to be within the normal limits, but functional hyperemia dynamics was not specifically evaluated (42). Previous fMRI data obtained using BOLD contrast in patients with probable cerebral amyloid angiopathy, who sometimes had severe clinical manifestations and cerebral hemorrhages, also cannot be compared with the present results (43). There are multiple strengths of our study. Patients were selected according to strict criteria; data were analyzed by mathematical modeling, allowing an independent evaluation of CBF dynamics along each task; measures were obtained in two different cortical areas using two different durations of stimulation; and results obtained in an initial group of patients were replicated in an independent sample. The limitations include the small spectrum of clinical manifestations, which prevented an analysis of clinical correlates; the variability of raw data, which reduced our ability to analyze results at the individual level; and the relative complexity of data processing pipelines and modeling.

In conclusion, the present study shows that NVC dynamics, which can be assessed in vivo using ASL-fMRI and long neural stimulations, is altered at the early stage of CADASIL. A late decrease in the cortical hyperemic response can be assessed using a simple marker calculated over a limited time-frame during 40-second neural stimulations. Additional studies are warranted to determine how these functional alterations evolve over time, correlate with disease severity, and can be used to monitor the progression of microvascular changes and treatment effects.