COMPOSITIONS AND METHODS OF TREATING AND PREVENTING NEURONAL DAMAGE FROM TRAUMATIC BRAIN INJURY

Governmental Interests

This invention was made with government support under grant no. NS072631 awarded by the National Institutes of Health. The Government has certain rights in the invention.

Background

Traumatic brain injury (TBI) is a leading cause of death and disability in children and young adults (Aitken et al., 2009; McCarthy et al., 2006). TBI results in neuronal death in the brain, including in the hippocampus and in the cortex. TBI can cause primary injury by immediate tissue disruption (Hall et al., 2004; Hall et al., 2005b; Singh et al., 2006), and also secondary injury of surviving cells triggered by the primary event (Faden et al, 1989; Fujimoto et al, 2004b; Hall et al., 2005b; Marion et al., 1997). Injury occurs in extensive brain areas, such as the cortex and hippocampus in the brains of both human beings (Ariza et al., 2006; Bigler et al., 1997; Isoniemi et al., 2006; Tate and Bigler, 2000; Umile et al., 2002; Wilde et al., 2007) and experimental animals (Bonislawski et al., 2007; Hall et al, 2005b; Lowenstein et al., 1992; Saatman et al., 2006a), leading to cognitive, sensory, and motor dysfunction (Greve and Zink, 2009; Hall et al., 2005b; Singh et al., 2006). There is no FDA approved therapeutic treatment for these disorders following TBI.

Following experimental traumatic brain injury (TBI), excitotoxicity is well documented and leads to cell death right after injury (Palmer et al., 1993). Excitotoxicity may cause cell death via either apoptosis or necrosis depending on the intensity of the initiating stimulus and the characteristics of the cell population (Ankarcrona et al., 1995; Bonfoco et al., 1995; Martin et al., 1998). Most of the neuronal death occurs within 24 hours post- TBI, and cell death continues at low level for at least another 2 weeks in a mouse TBI model (Zhou et al., 2012). The majority of the dying neurons did not exhibit morphological characteristics of apoptosis, and only a small subpopulation of the dying cells was positive for apoptotic markers. In contrast, most of the dying cells mainly died of necrosis, indicating that moderate traumatic brain injury mainly triggers rapid necrotic death.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors. It is active broadly in the adult brain, including the

hippocampus and cortex (Egan et al., 2003; Huang et al., 1999; Hyman et al., 1991). BDNF has diverse and important functions on neurons, such as helping survival of mature (Barde, 1994; Ghosh et al, 1994; Lindholm et al, 1996; Shulga et al, 2008) and immature neurons (Gao and Chen, 2009; Kirschenbaum and Goldman, 1995; Liu et al., 2003). BDNF is also the most abundant neurotrophin in hippocampal formation of both the adult rodent and human cortex (Maisonpierre et al., 1990; Timmusk et al., 1994;

Webster et al., 2002). When BDNF was conditionally knocked out in the hippocampus, reduction of BDNF expression exacerbated neuron death after TBI (Gao and Chen, 2009). This result suggests that BDNF is involved in regulating the neuronal survival, and potentially might be used to protect neurons from death following TBI. 7,8- dihydroxyflavone (DHF), a small molecule imitating BDNF, has been shown to protect wild-type neurons from apoptosis (Jang et al., 2010). Treatment with DHF improves neurological functions in rodents models of stress (Andero et al., 2012), depression (Liu et al., 2010), aging (Zeng et al., 2012a; Zeng et al., 2012b; Zeng et al., 2011), and Alzheimer's disease (Devi and Ohno, 2012).

Summary

Disclosed herein, DHF protects neuronal death, mainly necrosis, following TBI.

Treating TBI injured mice with DHF increased phosphorylation of Tyrosine-related kinase B (TrkB), which is a receptor of BDNF (Yan et al., 1997), and significantly reduced neuronal death in the hippocampus and tissue lesions in the cortex 24 hrs after

TBI. This indicated a neuroprotective effect of DHF following TBI. Further, inhibiting

BDNF signaling attenuated the neuroprotective effects of DHF.

The present invention includes methods and compositions for treating traumatic brain injury. In particular, the methods disclosed herein inhibit and/or prevent neuronal death subsequent to traumatic brain injury. In an embodiment, a method of treating TBI includes administering DHF or derivatives thereof to a subject subsequent to the occurrence of TBI. DHF or a derivative thereof may be administered in a clinical setting, wherein a subject is brought to the clinical setting subsequent to TBI. In an embodiment, DHF or a derivative thereof is administered to a subject at the site of injury through intraperitoneal (IP) injection.

In an embodiment, a method of treating TBI includes administering DHF or derivatives thereof prophylactically, whereby DHF or derivatives thereof are

administered prior to the occurrence of TBI. There are sports, jobs, etc. where the occurrence of TBI is likely. For instance, sports such as a football, soccer, boxing, hockey, martial arts, etc. where head trauma is likely to occur. In many instances, it is the repeated blows to the head that cause TBI. Thus, an embodiment includes administering DHF or a derivative thereof to an athlete prior and/or throughout a sporting activity.

In an embodiment, provided herein is use of DHF or derivatives thereof or a pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof in the manufacture of a medicament for preventing or inhibiting neuronal death subsequent to traumatic brain injury (TBI) prior to a likely occurrence of TBI or treating a subject following traumatic brain injury.

In an embodiment, provided herein is DHF or derivatives thereof or a

pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof for preventing or inhibiting neuronal death subsequent to traumatic brain injury (TBI) prior to a likely occurrence of TBI or treating a subject following traumatic brain injury.

In an embodiment, provided herein is a composition for preventing or inhibiting neuronal death subsequent to traumatic brain injury (TBI) prior to a likely occurrence of TBI or treating a subject following traumatic brain injury, comprising an effective amount of DHF or derivatives thereof or a pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof. In one aspect, the composition is a pharmaceutical composition or a beverage, for example, a sports drink. Brief Description of the Figures Figure 1 shows DHF protected neurons from death mediated by

glutamate/glycine-induced excitotoxicity in vitro, (a) Bright field image showing the cells treated with DMSO as control, (b) PI staining to detect dead cells treated with DMSO as control, (c) Merged image of panel (a) and panel (b). (d) Bright field image to show the cells treated with glutamate and glycine to induce excitotoxicity and pretreated with DMSO as control, (e) PI staining to detect dead cells treated with glutamate and glycine to induce excitotoxicity and pretreated with DMSO as control, (f) Merged image of panels (d) and (e). (g) Bright field image to show the cells treated with glutamate and glycine to induce excitotoxicity and pretreated with DHF. (h) PI staining to detect dead cells treated with glutamate and glycine to induce excitotoxicity and pretreated with DHF. (i) Merged image of panels (g) and (h). (j) Bright field image to show the cells treated with DHF but without induced excitotoxicity by glutamate and glycine, (k) PI staining to detect dead cells treated with DHF but without induced excitotoxicity by glutamate and glycine. (1) Merged image of panels (j) and (k). (m) Quantification of cell death treated by DMSO, glutamate/glycine, glutamate/glycine/DHF, or DHF. (n) Quantification of cell death treated by different concentration of DHF to assess the dose effect of DHF in neuroprotection after glutamate and glycine induced excitotoxicity.

Figure 2 shows DHF treatment reduced damage in the cortex following TBI. Mice were pretreated with DHF in DMSO or DMSO without DHF as control before receiving TBI surgery, (a) Epicenter section of the brain pretreated with DMSO without DHF was stained with cresyl violet, (b) Epicenter section of the brain pretreated with DHF was stained with cresyl violet, (c) Percentage of cortical lesion in the ipsilateral cortex compared with the contralateral cortex.

Figure 3 shows DHF treatment reduced cell death in the hippocampus following TBI. FJB staining was performed to detect dead cells in the hippocampus at 24 hours after TBI surgery, (a) Brain section containing hippocampal dentate gyrus from mice that received sham surgery. Section was counter staining with DAPI (4',6-diamidino-2- phenylindole) to show the cell nuclei in blue, (b) FJB staining to detect FJB-positive dead cells (in green) in the hippocampal dentate gyrus from TBI injured mice, but not treated with DHF. The section was counter staining with DAPI to show the cell nuclei in blue, (c) FJB staining to detect FJB-positive dead cells (in green) in the hippocampal dentate gyrus from TBI injured mice with DHF pretreatment. The section was counter staining with DAPI to show the cell nuclei in blue, (c) FJB staining to detect FJB-positive dead cells (in green) in the hippocampal dentate gyrus from TBI injured mice with DMSO pretreatment. Section was counter staining with DAPI to show the cell nuclei in blue, (e) Quantification of FJB-positive dead cells in the different subregions of the hippocampus.

Figure 4 shows DHF treatment increased the number of newborn immature neurons in the hippocampus following TBI. Immnunostaining with antibody against Dcx was performed to exhibit newborn immature neurons in the hippocampal dentate gyrus of mice 24 hours after surgery, (a) Dcx-positive newborn immature neurons (in red) in the hippocampal dentate gyrus of sham-operated mouse. Section was counter staining with DAPI to show the cell nuclei in blue, (b) Dcx-positive newborn immature neurons (in red) in the hippocampal dentate gyrus of TBI injured mouse. Section was counter staining with DAPI to show the cell nuclei in blue, (c) Dcx-positive newborn immature neurons (in red) in the hippocampal dentate gyrus of TBI injured mouse pretreated with DHF. Section was counter staining with DAPI to show the cell nuclei in blue, (d) Dcx-positive newborn immature neurons (in red) in the hippocampal dentate gyrus of TBI injured mouse and pretreated with DMSO. Section was counter staining with DAPI to show the cell nuclei in blue.

Detailed Description

Definitions

The term "in combination with" refers to administration of one or more therapeutic agents in addition to DHF or derivatives thereof, and includes simultaneous (concurrent) and consecutive administration in any order.

The term "7, 8-dihydroxyflavone" or "DHF" refers to 7,8-dihydroxy-2-phenyl- 4 7-l-benzopyran-4-one, which is a compound of the formula DHF is a tyrosine kinase receptor B (TrkB) agonist that binds to the extracellular domain of the receptor (¾ = 320 nM), provoking receptor dimerization and autophosphorylation.

The term "mammal" for purposes of treatment or therapy refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, and the like. Preferably, the mammal is human.

The term "treatment" or "treating" is an approach for obtaining beneficial or desired clinical results. For purposes herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. For the purposes herein, "treatment" does not include "preventing" or "prevention" or "prophylaxis".

The term "compound" refers to DHF or derivatives thereof or a pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof, and also includes protected derivatives thereof. Compounds may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. According to this invention, the chemical structures depicted herein, including the compounds of this invention, encompass all of the corresponding compounds' enantiomers, diastereomers and geometric isomers, that is, both the stereochemically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and isomeric mixtures (e.g., enantiomeric, diastereomeric and geometric isomeric mixtures). In some cases, one enantiomer, diastereomer or geometric isomer will possess superior activity or an improved toxicity or kinetic profile compared to other isomers. In those cases, such enantiomers, diastereomers and geometric isomers of compounds of this invention are preferred.

The term "polymorph" refers to a solid crystalline form of a compound disclosed herein or complex thereof. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermo dynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of polymorphs can affect their processing. For example, one polymorph might be more likely to form solvates or might be more difficult to filter or wash free of impurities than another due to, for example, the shape or size distribution of particles of it.

The term "hydrate" refers to a compound disclosed herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non- covalent intermolecular forces.

The term "clathrate" refers to a compound disclosed herein or a salt thereof in the form of a crystal lattice that contains spaces (e.g., channels) that have a guest molecule (e.g., a solvent or water) trapped within.

The term "prodrug" refers to a drug molecule of a compound of the invention that is biologically inactive until it is activated by a metabolic process. A prodrug includes a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of the invention. Prodrugs may become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds disclosed herein that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Other examples of prodrugs include derivatives of compounds disclosed herein that comprise—NO, --NO ₂ , --ONO, or --ONO ₂ moieties. Prodrugs can typically be prepared using well known methods.

The term "occurrence likely to produce TBI" refers to an event, situation, or happenstance where traumatic brain injury is likely to occur. An occurrence likely to produce can be while playing a sport. For example, TBI is likely to occur to during football due to the collisions involving the head. Playing the sport includes both games and practice. Another occurrence likely to produce TBI is combat operations. Another occurrence likely to produce TBI includes older persons with an unsteady gait in general circumstances.

Traumatic Brain Injury

At the most basic definition, traumatic brain injury (TBI) is simply damage to the brain due to injury. TBI refers to both penetrating and non-penetrating injury. A non- penetrating traumatic injury can include a blow to the head whereby the brain is injured as it impacts with the inside of the skull one or more times. The blow can result from falls (e.g., falling down stairs, an older person falling in the bathroom), vehicle collisions, sports collisions (e.g., football, soccer (e.g., headers), hockey (e.g., checking), boxing, martial arts, lacrosse, skateboarding, etc.), and combat (e.g., explosive blasts, hand-to- hand combat, etc.).

TBI can result in mild to severe damage. Mild damage may only cause temporary dysfunction of cells. However, severe damage can result in tearing of tissues, bleeding, and neuronal death. Primarily, TBI causes cell death in the cerebral cortex and secondarily in the hippocampus (Fujimoto et al., 2004a; Hall et al., 2005a; Saatman et al., 2006b). This cellular death, including neuronal cell death, is necrotic death. Dead cells subsequent to TBI distinctly bear the hallmarks of necrosis and not apoptosis. TBI and neuronal death can lead to cognitive deficits (e.g., memory, learning, etc.), sensory deficits (e.g., vision, hand-eye coordination, etc.), communicative disorders (e.g., dysarthria, aphasia, etc.), and behavioral/emotional disorders (e.g., depression, anxiety, irritabi lity , etc . ) .

Methods of Administering DHF

In an embodiment, a method of treating TBI includes administering DHF or derivatives thereof to a subject subsequent to the occurrence of TBI. DHF or a derivative thereof may be administered in a clinical setting, wherein a subject is brought to the clinical setting subsequent to TBI. In an embodiment, DHF or a derivative thereof is administered to a subject at the site of injury. The site of injury can be in one's home, a place of business, at a sporting event, or the battlefield.

In an embodiment, a method of treating TBI includes administering DHF or derivatives thereof prophylactically, whereby DHF or derivatives thereof are

administered prior to the occurrence of TBI. There are sports, jobs, etc. where the occurrence of TBI is likely. For instance, sports such as a football, soccer, boxing, hockey, martial arts, etc. where head trauma is likely to occur. In many instances, it is the repeated blows to the head that cause TBI. Thus, an embodiment includes administering DHF or a derivative thereof to an athlete prior to a sporting activity. DHF or a derivative thereof can be administered to an athlete prior to a game (or match) or practice where a head injury is likely to occur. Likewise, members of the military have been frequently exposed to explosive devices in recent conflicts. Infantry can be administered DHF or derivatives thereof prior to a patrol, convoy, etc., or any combat operations in general.

In addition, DHF or derivatives thereof are administered in combination or alternation with one or more additional compounds. TBI, especially blunt force trauma, can cause inflammation. In an embodiment, DHF or a derivative thereof can be administered in combination with a diuretic, which is administered to reduce intra-cranial pressure. Diuretics include, but are not limited to, furosemide, hydrochlorothiazide, metalozone, eplerenone, etc. For the first couple of weeks, humans are at increased risk for seizures following moderate to severe TBI. In an embodiment, DHF or a derivative thereof can be administered in combination with an anti-seizure therapeutic. Examples of anti-seizure therapeutics include, but are not limited to, gabapentin, carbamazepine, phenytoin, oxcarbezepine, divalproex sodium, clonazepam, topiramate, and valproic acid. Pharmaceutically Acceptable Salts

In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compound as a pharmaceutically acceptable salt may be appropriate. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. In particular, examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be formed, including, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard, well known procedures art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

Compositions

Compositions based DHF or a pharmaceutically acceptable salt, can be prepared in a therapeutically effective amount for any of the indications described herein, in combination with a pharmaceutically acceptable additive, carrier or excipient. The therapeutically effective amount may vary with the condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used, as well as the patient treated.

In one aspect, DHF is formulated preferably in admixture with a pharmaceutically acceptable carrier. In general, it is preferable to administer the pharmaceutical composition in orally administrable form, but formulations may be administered via parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository or other route. Intravenous and intramuscular formulations are preferably administered in sterile saline. One of ordinary skill in the art may modify the formulation within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising its therapeutic activity. In particular, a modification of a desired compound to render it more soluble in water or other vehicle, for example, may be easily accomplished by routine modification (salt formulation, esterification, etc.). The amount of compound included within a therapeutically active formulation is an effective amount for treating neuronal death in TBI. In general, a therapeutically effective amount of the present compound in pharmaceutical dosage form usually ranges from about 0.1 mg/kg to about 100 mg kg or more, depending upon the compound used, the degree of TBI, and the route of administration. For purposes of the present invention, a prophylactically or preventively effective amount of the compositions, according to the present invention, falls within the same concentration range as set forth above for therapeutically effective amount and is usually the same as a therapeutically effective amount.

Administration of DHF or derivatives thereof may range from continuous

(intravenous drip) to several oral administrations per day (for example, Q.I.D., B.I.D., etc.) and may include oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancement agent), buccal and suppository administration, among other routes of administration. Enteric-coated oral tablets may also be used to enhance bioavailability and stability of the compounds from an oral route of administration. The most effective dosage form will depend upon the pharmacokinetics of the particular agent chosen, as well as the severity of TBI in the patient. Oral dosage forms are particularly preferred, because of ease of administration and prospective favorable patient compliance.

To prepare the pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably mixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. If desired, the tablets or capsules may be enteric-coated for sustained release by standard techniques. The use of these dosage forms may significantly impact the bioavailability of the compounds in the patient.

For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients, including those that aid dispersion, also may be included. Where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

In embodiments, compounds and compositions are used to treat, prevent or delay the onset of neuronal death related to TBI. Preferably, to treat, prevent or delay the onset of neuronal death, compositions are administered in oral dosage form in amounts ranging from about 250 micrograms up to about 1 gram or more at least once a day, or up to four times a day. Compounds are preferably administered orally, but may be administered parenterally, topically or in suppository form.

In an embodiment, DHF or derivatives thereof can be formulated as part of a beverage, including a sports drink (e.g., Gatorade ), energy drink (e.g., Red Bulr), nutritional drink (e.g., Ensure ^® ) or any type of beverage that replenishes or supplements vitamins, electrolytes, and the like. Such drinks can help prevent or inhibit neuronal death due to TBI from sports injury, combat, or other event. Embodiments include consuming or administering a beverage comprising DHF or a derivative thereof to a subject before, after, or throughout an occurrence likely to produce TBI.

EXAMPLES

Example 1: DHF protected neurons against excitotoxic injury in vitro

This example is to determine the neuroprotective effect of DHF against excitotoxic injury and its optimal dose on the cultured neurons in vitro.

Methods Hippocampal cell culture and in vitro TBI modeling. Brains of the C57BL/6 mice (post-natal day 0 (P0)) were dissected, the meninges were removed, and the hippocampi were isolated. Hippocampal tissue was digested with papain, and single-cells were collected, plated onto polylysine-coated coverslips in plates and maintained in a B27 supplemented Neurobasal™ serum-free medium (Brewer et al., 1993). To imitate in vivo traumatic injury, the culture at division (DIV 5) was exposed to 100 μΜ glutamate with 20μΜ glycine or same volume of DMSO as control for 60 min and then rested for 24 hrs.

DHF treatment. Thirty minutes before glutamate and glycine treatment, cells were treated with DHF in DMSO at a final concentration of 500 nM or DMSO without DHF as control.

Propidium iodide (PI) staining and cell counting. PI (10μg/mL) was applied to cell culture at 24 hours after treatment. Following 30 min at 37°C in a humid incubator, cells were washed briefly with PBS and then fixed with 2% paraformaldehyde (PFA) in PBS. Fixed cells were checked by phase contrast or fluorescent microscopy. Five random fields on each coverslip were chosen to take images of both bright field and PI stain at a magnification of 20X using an inverted microscopy system (Zeiss Axiovert 200 M equipped with Apotome) interfaced with a digital camera (Zeiss Axio Cam M c5) controlled by software (AxioVision, v4.0). Captured images were assembled and labeled in Photoshop 7.0 (Adobe Systems). The numbers of the cells and those PI positive cells were counted and the percentages of PI positive cells in total cells were calculated. Triplicate slides were included in each experimental group.

Statistical analyses. All data are presented as mean ± standard error (SE), with the number of repetitive experiments or mice indicated. Mean values were statistically compared using student's t-test or one-way ANOVA followed by Tukey's post hoc testing.

Results

To assess the effect of DHF on neuronal survival following the glutamate/glycine- induced injury, DHF in DMSO in concentrations of 500 nM and DMSO without DHF were administrated into the culture media 30 min before injury. Twenty-four hours after treatment, PI staining was performed to detect the dead cells. In the vehicle (DMSO) treated group, 6.45% ± 1.06% of neurons were labeled by PI (Figure la-c and m).

Glutamate/glycine caused massive neuronal death (46.92% ± 2.53%, P=0.0001 vs.

vehicle group) (Figure 1 d-f and m). DHF treatment significantly reduced the Pl-positive cells to 20.15% ± 1.09%, suggesting DHF affectively protected 57.05%> of neurons from death induced by Glutamate/Glycine (Figure 1 g-I and m). Short-term treatment of DHF did not significantly affect the death (6.56% ± 1.28%) of healthy neurons without glutamate/glycine treatment (Figure 1 j-1 and m). These results indicate that DHF protects neurons from death induced by glutamate/glycine-mediated excitotoxicity in vitro.

To determine an optimal dose of DHF in neuroprotection, the neurons at DIV 5 were incubated with a different concentration of DHF (from 0 to 5000 nM (Figure 1 n)) 30 minutes before glutamate/glycine treatment. Twenty-four hours after treatment, PI staining was performed to detect the dead neurons. Without DHF treatment, 46.92% ± 2.53% of neurons stained positive for cell death. Even at a low concentration of DHF (10 nM), cell death was reduced to 37.11% ± 1.33%, indicating that DHF exhibited neuroprotective effects at a low concentration. Neuroprotection was concentration dependent. As the concentration of DHF increased, neuroprotection increased. The rate of cell death induced by Glutamate/glycine-mediated excitotoxicity decreased to 23.13% ± 3.28% when treated with DHF at a concentration of ΙΟΟηΜ, 17.05% ± 1.96% at 500 nM. However, the neuroprotective effect reached a plateau after a concentration of 500 nM. The rate of cell death was 14.26% ± 1.52% when treated with DHF at a concentration of 1.0 μΜ; 12.72% ± 0.57% at 5.0 μΜ. A DHF concentration more than 500 nM did not further reduce the rate of cell death, thereby suggesting that 500 nM is an optimal concentration for neurprotection in vitro.

Further, neurons displayed healthy morphologies with round-shaped cell body and appropriate processes without swelling or retraction when treated with high doses of DHF (e.g., 5.0 μΜ, which is 10 times higher in concentration than the optimal dose) (data not shown). These data suggest that DHF has very low cellular toxicity. Example 2: DHF reduced cortex lesions after TBI DHF is a small molecule (MW = 254.25 g/mol) with the ability to penetrate through the blood brain barrier and reach the brain areas affected after TBI (Andero et al., 2011). DHF was administered with intraperitoneal injection to test whether DHF administration could reduce neuronal death in the cortex after TBI in vivo.

Methods

Animal care. Male C57BL/6 mice (The Jackson Laboratories, Bar Harbor, ME) were group housed with a 12/12-hr light/dark cycle and had access to food and water ad libitum. The mice 8-10 weeks of age at the time of the experiments. All procedures were performed under protocols approved by the Animal Care and Use Committee of Indiana University.

Traumatic brain injury (TBI) model. C57BL/6 male mice, 8-10 weeks old, were subjected to moderate controlled cortical impact (CCI) injury, as we previously described (Gao et al., 2011) by using an electromagnetic device (Brody et al., 2007). Briefly, the mice were anesthetized with avertin and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA) prior to TBI. Using sterile procedures, the skin was retracted and a 4 mm craniotomy centered between the lambda and bregma sutures was performed. A point was identified midway between the lambda and bregma sutures and midway between the central suture and the temporalis muscle laterally. The skullcap was carefully removed without disruption of the underlying dura. Prior to the injury, the impacting tip was angled perpendicularly to the exposed cortical surface, and then the moderate severity brain injury was introduced with the depth of deformation set at 1.0 mm and the piston velocity controlled at 3.0 m/sec. Sham (non- injured) animals received the craniotomy, but no CCI injury. During all surgical procedures and recovery, the core body temperature of the animals was maintained at 36-37°C.

DHF or control treatment. One hour before surgery, the mice were treated either with DHF in DMSO (5mg/kg, i.p.) or same volume of DMSO without DHF (i.p.) as control.

Tissue processing. Twenty-four hours after surgery, animals were deeply anesthetized with an overdose of avertin and then perfused transcardially with 0.9% saline, followed by an ice-cold fixative containing 4% PFA in PBS. The brains were removed and postfixed in 4% PFA overnight, then cryoprotected with 30% sucrose for 48 hr. Serial coronal sections (30 μιη thick) were cut using a cryostat (Microm HM 500 M) and stored at -20°C.

Cortex lesion measurement. In order to evaluate the spare cortex, one-in-six brain sections from a series of 30 μηι thick cross sections of whole brains (180 μηι apart) were selected to stain with cresyl violet (Gao and Chen, 201 1). The boundary contours of the contralateral and ipsilateral spare cortex were drawn with an Olympus BX60 microscope attached to a Neurolucida ^® system (Microbrightfield Inc., Colchester, VT). The three- dimensional reconstruction was done to measure the enclosed volume within the contours with the automated software NeuroExplorer ^® (Nex Technologies, Littleton, MA). The cortex lesion was determined by measuring the volume of cavity in the cortex. The degree of cortex lesion is represented as percentage of cortex loss in volume, calculated with the following formula: [(total volume of contralateral cortex - total volume of spared ipsilateral cortex) / total volume of contralateral cortex] x 100%.

Statistical analysis. All data are presented as mean ± standard error (SE), with the number of repetitive experiments or mice indicated. Mean values were statistically compared using student's t-test or one-way ANOVA followed by Tukey's post hoc testing. Results

Although the sham surgery mice were treated with either DHF or DMSO, they did not show any cortex cavities at either the control lateral or ipsilateral cortex. Whereas, the TBI-injured mice that received DMSO treatment as control lost 16.75% ± 1.46% % of its cortex at the ipsilateral side at 24 hours after injury (Figure 2a), and there was not a lesion cavity found at the contralateral cortex (Figure 2a). The TBI-injured mice that received DHF treatment had reduced lesions to 12.43% ± 0.74%o of its cortex (Figure 2b), which is 25.8% decrease in tissue lesion compared to the control treated group. The reduction in cortex lesions with DHF treatment was statistically significant. These data indicate that DHF treatment provides a neuroprotective effect against TBI (Figure 2c).

Furthermore, obvious behavioral changes were not observed between the control treated and DHF treated animals in a) the length of time to wake up following anesthesia, b) in motor function, and c) pain reaction. These observations suggest a lack of side effects in vivo.

Example 3: DHF reduced immature neuron death and increased the number of newborn immature neurons in the hippocampus after TBI

After the effect of DHF in hippocampus had been identified, the effect in the cortex was also explored. To examine the effect of DHF on the tissue loss in the cortex, the area of cortical cavity was assessed after staining of the sections with cresyl violet.

Methods

Animals were cared for as described in Example 2.

Traumatic brain injury (TBI) was performed as described in Example 2.

Animals were treated with DHF or control treatment as described in Example 2. Briefly 1 hour before surgery, the mice were treated either with DHF in DMSO (5 mg/kg i.p.) or same volume of DMSO without DHF (i.p.) as control.

Tissue processing was performed as described in Example 2. Briefly, 24 hours after surgery, animals were deeply anesthetized with an overdose of avertin and then perfused transcardially with 0.9% saline, followed by an ice-cold fixative containing 4% PFA in PBS. The brains were removed and postfixed in 4% PFA overnight, then cryoprotected with 30% sucrose for 48 hr. Serial coronal sections (30 μηι thick) were cut using a cryostat (Microm HM 500 M) and stored at -20°C.

FJB staining of dying neurons. The staining procedures were implemented as described previously (Gao and Chen, 2009, 201 1; Schmued and Hopkins, 2000). Briefly, the sections mounted on slides were incubated in 0.06% potassium permanganate for 20 min and rinsed in distilled water for 5 min. Thereafter the sections were incubated in 0.0004% FJB (Histo-Chem Inc., Jefferson, AR) for 20 mm and counterstained with DAPI for 5 min. Finally, sections were rinsed in distilled water and air dried overnight. The dry slides were mounted with DPX Mounting Medium (Fluka, Milwaukee, Wi).

Hippocampal neuron death assessment. To determine the FJB-positive cells density in hippocampus across the whole hippocampus, one section out of every six throughout the entire extent of the hippocampal formation was selected for assessment. The anatomical boundaries of each hippocampal subregion (CA1 ; CA3; granular cell layer, GCL; molecular layer, ML; Hilus) were identified as described before (Amaral and Witter, 1989). The number of FJB-positive neurons in each subregion was determined by a blinded quantitative histological analysis through the Z-axis under a microscope system (Zeiss Axiovert 200 M, Carl Zeiss Microimaging, Inc., Thornwood, NY) with a 40X objective. The subregion area (2 μηι) was measured with an imaging software

(AxioVision, v4.0, Carl Zeiss Microimaging, Inc.). The results of FJB-positive cells density were calculated with the section thickness (30 μηι) and presented as N/mm ³ .

Immunohistochemistry. Tissue sections were rinsed with PBS and then blocked with 10% donkey serum in PBS with 0.3% Triton ^® for 2 hours at 4°C. The first antibodies (sheep-anti-mouse p-TrkB (phospho Y816), 1 :20, ab74841, Abeam; mouse- anti-mouse NeuN, 1: 100, MAB377, Chemicon; guinea pig-anti-mouse double cortin (DCX), 1 : 1000, Millipore, ab2253) were applied and incubated for 48 hours at 4°C. After rinsing with PBS, the sections were incubated with the donkey anti-sheep secondary antibody (Alexa488 IgG, 1 :1000, Al 1015, Invitrogen) in 10% donkey serum in PBS for 1 hour at 4 degrees then rinsed. The sections were also incubated with the goat-anti-mouse (cy3 IgG, 1 :400, A10521 , Invitrogen) and goat-anti-guinea pig (cy5 IgG, 1 :200, A21450, Invitrogen) secondary antibodies in blocking buffer (1% bovine serum albumin, 5% normal goat serum in PBS) for 1 hour at 4°C, counterstained with DAPI for 2 min, and then rinsed with PBS. The sections were rinsed briefly with water and mounted on slides with the mounting medium. Images were taken using a microscope.

Statistical analyses. All data are presented as mean ± standard error (SE), with the number of repetitive experiments or mice indicated. Mean values were statistically compared using student's t-test or one-way ANOVA followed by Tukey's post hoc testing.

Results

DHF in DMSO (5 mg/kg, i.p. injection) or DMSO without DHF was administered one hour before the TBI insult, and animals were sacrificed 24 hours after TBI. Brain sections were selected to evaluate cell death with FJB staining. The dying or dead neurons were stained by FJB in green. There was no FJB-positive cell observed in the dentate gyras of sham-operated animals (Figure 3a). In contrast, the FJB-positive cells were observed in the hippocampus of mice in all groups, except sham control. Most of the FJB-positive cells were located at the inner one-third portion of the dentate gyras of TBI inured hippocampus (Figure 3b), where the adult-born immature neurons locate. Previous studies showed that the majority of those FJB-positive cells are immature neurons (Gao, 2008), and most of the cells die of necrosis (Zhou, 2012). Quantification showed that the density of FJB positive cells in the dentate gyrus was 25,395 ± 2598/mm ³ (Figure 3d). The amount of FJB-positive cells in the dentate gyrus of TBI injured animals was statistically significantly less after DHF treatment. Quantification showed that the density of FJB positive cells in the dentate gyrus of DHF treated animals decreased to

19,505 ± 600/mm (Figure 3d), representing a decrease in cell death of 23.2% . Treatment with DMSO as a control did not significantly affect cell death in the hippocampal dentate gyrus (Figure 3d). These data indicate that DHF protected against neuronal death in the hippocampal dentate gyrus following TBI. The majority of the neurons were immature granule neurons.

Although most of the FJB-positive cells were found at the hippocampal dentate gyrus, there were small amounts of neuronal death also observed in other hippocampal subregions including CA1 , CA3, and the hilus. The densities of FJB-positive cells in these areas were 233 ± 146/mm ³ (CA1), 3863 ± 1045/mm ³ (CA3), and 1643 ± 771/mm ³ (hilus) in the controlled treated hippocampus. In the hippocampuses of DHF treated animals, the densities of FJB-positive cells in these areas were slight changes to 308 ± 53/mm (CAl), 3808 ± 504/mm ³ (CA3), and 1257 ± 226/mm ³ (hilus).

To further determine the neuroprotective effect of DHF in immature neurons following TBI, DHF treatment of the immature neurons in the hippocampal dentate gyrus was evaluated. Brain sections from mice 24 hours after TBI or sham-operated mice were used to assess the newborn immature neurons in the hippocampal dentate gyrus. A brain section at the epicenter was selected to immunostain with antibody against Dcx, a marker for newborn immature neurons ( orzhevskii et al., 2006; Mullen et al., 1992). The newborn immature neurons at the dentate gyrus were stained in red (Figure 4). There were 187 ± 14 immature neurons at the epicenter section containing the dentate gyrus of sham-operated mice (Figure 4a and 4e). At 24 hours after TBI, this number decreased to 76 ± 4 without any treatment (Figure 4b and 4e), representing a 59.4% reduction. Whereas, the number of newborn immature neurons was 142 ± 9 in the DHF treated animals (Figure 4c and 4e), a reduction of 24.1%. The difference in cell death was 35.3% survived with DHF treatment that would have died, mainly from necrosis, without DHF treatment. These data indicate that one single injection of DHF protected newborn immature neurons from death following TBI.