Neuroprotection after traumatic brain injury in heat-acclimated mice involves induced neurogenesis and activation of angiotensin receptor type 2 signaling

Long-term exposure of mice to mild heat (34°C±1°C) confers neuroprotection against traumatic brain injury (TBI); however, the underling mechanisms are not fully understood. Heat acclimation (HA) increases hypothalamic angiotensin II receptor type 2 (AT₂) expression and hypothalamic neurogenesis. Accumulating data suggest that activation of the brain AT₂ receptor confers protection against several types of brain pathologies, including ischemia, a hallmark of the secondary injury occurring following TBI. As AT₂ activates the same pro-survival pathways involved in HA-mediated neuroprotection (e.g., Akt phosphorylation, hypoxia-inducible factor 1α (HIF-1α), and brain-derived neurotrophic factor (BDNF)), we examined the role of AT₂ in HA-mediated neuroprotection after TBI. Using an AT₂-specific antagonist PD123319, we found that the improvements in motor and cognitive recovery as well as reduced lesion volume and neurogenesis seen in HA mice were all diminished by AT₂ inhibition, whereas no significant alternations were observed in control mice. We also found that nerve growth factor/tropomyosin-related kinase receptor A (TrkA), BDNF/TrkB, and HIF-1α pathways are upregulated by HA and inhibited on PD123319 administration, suggesting that these pathways play a role in AT₂ signaling in HA mice. In conclusion, AT₂ is involved in HA-mediated neuroprotection, and AT₂ activation may be protective and should be considered a novel drug target in the treatment of TBI patients.     

Quantitative autoradiography of angiotensin II AT2 receptors with [I]CGP 42112

Most radiolabeled ligands for angiotensin II (Ang II) receptors do not discriminate between the AT₁ and AT₂ receptor subtypes, which must be distinguished by displacement with selective AT₁ or AT₂ ligands. We compared [¹²⁵I]CGP 42112 with the non-selective agonist [¹²⁵I]Sar¹Angiotensin II. We studied the inferior olive, medial geniculate nucleus and the adrenal medulla, areas rich in AT₂ receptors, using both ligands with quantitative autoradiography and membrane binding techniques. [¹²⁵I]CGP 42112 bound with high affinity (K_d = 0.07−0.3nM, depending on the area studied). [¹²⁵I]CGP 42112 binding was selective for AT₂ receptors, as determined by lack of competition with the AT₁ ligand losartan, and competition by the AT₂ ligands PD 123177 and unlabeled CGP 42112 and the non-selective peptides Ang II and angiotensin III (Ang III). Using [¹²⁵I]CGP binding, we found the same order of potency: CGP 42112 > Ang II = Ang III > PD 123177 using both quantitative autoradiography or membrane binding methods. Our results demonstrate that [¹²⁵I]CGP 42112 is the most selective, highest affinity ligand available for AT₂ receptors. Because of these characteristics, and low non-specific binding, quantitative autoradiography with [¹²⁵I]CGP 42112 is the method of choice to selectively characterize AT₂ receptors, especially in tissues like the brain, with a highly heterogeneous distribution of receptor subtypes.     

Expression of non-angiotensin II [I]CGP 42112 binding sites on activated microglia after kainic acid, induced neurodegeneration

[¹²⁵I]CGP 42112, first developed to identify angiotensin II receptor subtype 2 (AT₂), was recently shown to bind to a novel non-angiotensin binding site in injured rat brain tissue. We addressed the question whether non-angiotensin [¹²⁵I]CGP 42112 binding appears after kainic acid induced hippocampal neurodegeneration, a process of neuronal cell death at a distance from the toxin injection site. After intraventricular kainic acid injection, we found non-angiotensin [¹²⁵I]CGP 42112 binding in the hippocampal areas CA3 (4 and 14 days after injection), CA1 and CA4 and the subiculum (14 days after injection). In addition, 14 days after kainic acid injection, [¹²⁵I]CGP 42112 binding was found in 50% of the animals, in the thalamus, amygdala and piriform cortex, areas receiving projections from the hippocampus and suffering kainic acid induced delayed neurodegenaration. The loss of neurons in these regions was accompanied by an accumulation of activated microglia as demonstrated by immunostaining with the specific antibodies OX-42 and ED1. The time course and regional pattern of OX-42/ED1 positive immunostaining was identical with the appearance and distribution of the non-angiotensin [¹²⁵I]CGP 42112 binding site. The non-angiotensin [¹²⁵I]CGP 42112 binding was not detected in brain regions unaffected by kainic acid injection. Our findings indicate the expression of a novel [¹²⁵I]CGP 42112 binding site on activated microglia. This site appears at a distance from the lesion and may be of importance in the process of neuronal death and brain tissue repair.     

Angiotensin AT1 receptors in the preoptic area negatively modulate the cardiovascular and ACTH responses induced in rats by intrapreoptic injection of prostaglandin E2

We previously reported that brain angiotensin II type 2 (AT₂) receptors contribute to the hyperthermia induced by intrahypothalamic (intrapreoptic (i.p.o.)) administration of prostaglandin E₂ (PGE₂) in rats. The present study was carried out to investigate the role of angiotensin II (ANG II) receptors in the cardiovascular and adrenocorticotropic hormone (ACTH) responses induced in rats by i.p.o. injection of PGE₂. PGE₂ (100 ng) produced marked increases in blood pressure, heart rate, and plasma ACTH concentration. These changes were significantly enhanced by i.p.o. treatment with an AT₁-receptor antagonist, losartan, while an AT₂-receptor antagonist, CGP 42112A, had no effect. In contrast, losartan, but not CGP 42112A, reduced the pressor and ACTH responses to i.p.o. injection of a large dose of “exogenous” ANG II (25 ng). These results suggest that while “endogenous” ANG II exerts inhibitory effects on both the cardiovascular and the ACTH responses to i.p.o. PGE₂ by way of preoptic AT₁-receptors, a large dose of exogenous ANG II produces effects opposite to those induced by the endogenous ANG II that is released locally and in small amounts by i.p.o. PGE₂.     

Expression of a novel angiotensin II receptor subtype in gerbil brain

Angiotensin II receptors are highly localized in adult gerbil brain. Apparent receptor number is high in subfornical organ, vascular organ of the lamina terminalis, nucleus of the solitary tract, hippocampus, and in the anterior pituitary gland. In the hippocampus, binding is localized to the stratum oriens, radiatum, the lacunar molecular layers of the CA1 subfield, and the molecular layer of the gyrus dentatus, with a medial to lateral and anterior to posterior gradient in receptor expression. Binding is absent from the pyramidal layer of the CA1 subfield and from the granular cell layer of the gyrus dentatus, areas rich in angiotensin IV binding. Characterization in the hippocampus revealed the presence of a high affinity receptor, sensitive to incubation with the guanine nucleotide GTPγS, and displaced by angiotensin II = angiotensin III < Sar¹-Ile⁸-angiotensin II, but not by angiotensin IV or other angiotensin fragments, the AT₁ receptor antagonist losartan, or the AT₂ ligands CGP 42112 or PD 123177. In other brain areas, binding was equally insensitive to displacement by AT₁ or AT₂ ligands, with the exception of binding in the olfactory bulb, which was totally displaced by CGP 42112 and PD 123177, but not by losartan. In the gerbil, most of the brain and pituitary angiotensin II receptors are different from the AT₁ AT₄ and AT₄ subtypes, and should be considered ‘atypical’ until further characterization.     

Angiotensin II receptor subtypes and phosphoinositide hydrolysis in rat adrenal medulla

Angiotensin 11 (ANG) receptor subtypes were characterized by quantitative autoradiography after incubation with the ANG agonist [¹²⁵I]Sar¹-ANG in rat adrenal medulla. ANG receptors are highly localized in adrenal medulla. Specific binding was displaced by 4% and by 95% with the AT₁ receptor blocker losartan and the AT₂ receptor competitor CGP 42112A, respectively. Analysis of competition curves indicated relative binding potencies for the AT2 population of CGP 42112A>PD 123319> PD 123177. ANG stimulated inositol phosphate formation in a dose-dependent manner in rat adrenal medulla. Losartan at concentrations of 10⁻⁹ to 10⁻⁵ M antagonized the effect of ANG, whereas PD 123177 or PD 123319 had no antagonistic action. However, at a higher concentration (10⁻⁵ M) PD 123177 or PD 123319 potentiated the effect of ANG on InsP₁-accumulation. In the presence of PD 123319 (10⁻⁵ M) ANG dose-response curve was shifted to the left with no change in the maximal effect. This affect was blocked by the addition of losartan (10⁻⁵ M). On the contrary, the addition of CGP 42112A (10⁻⁵ M) inhibited ANG-induced increase in InsP_i-accumulation. On the other hand, ANG and CGP 42112A reduced basal cyclic GMP formation, this effect was partially reverted by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. Our results further demonstrate the presence of two ANG receptor subtypes in adrenal medulla: ANG binding to AT₁ receptor stimulates inositol phospholipid metabolism, whereas ANG binding to AT₂ receptors decreases both inositol phosphate production and cGMP formation.     

Angiotensin II receptor subtypes and angiotensin-converting enzyme in the fetal rat brain

Angiotensin II (ANG II) receptor subtypes (AT₁, displaced by losartan, and AT₂, displaced by CGP 42112A) were characterized by quantitative autoraography after incubation with the ANG II agonist [¹²⁵I]Sar¹-ANG II, in specific brain nuclei of 19-day-old rat embryos. Binding to AT₁ receptors, located in the subfornical organ, paraventricular nucleus, nucleus of the solitary tract and choroid plexus, was sensitive to incubation with GTPγS. The sensitivity of AT₂ receptors to GTPγS was heterogeneous. In the ventral thalamic, rostral hypoglossal and medial geniculate nuclei, and in the locus coeruleus, binding to AT₂ receptors was sensitive to GTPγS and these areas belong to the AT_2A subgroup. Conversely, in the inferior olive, medial (fastigial) cerebellar nucleus and caudal part of the hypoglossal nucleus, areas belonging to the AT_2B subgroup, binding was insensitive to GTPγS. AT₂ receptors were also present in cerebral arteries. In the fetal anterior pituitary, AT₁ receptors predominated. The angiotensin-converting enzyme (ACE; EC 3.4.15.1) was studied by autoradiography with the selective inhibitor [¹²⁵I]351A. In 19-day-old embryos, ACE was highly expressed in chroid plexus, with high concentrations in subfornical organ, posterior pituitary and cerebral arteries. No ACE binding was detected in extrapyramidal structures or anterior pituitary in 19-day-old embryos.     

Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [¹²⁵I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [¹²⁵I]Sar¹–Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [¹²⁵I]Sar¹–Ang II binding was displaced by the AT₁ receptor antagonist losartan, indicating the presence of AT₁ receptors. In the paraventricular nucleus [¹²⁵I]Sar¹–Ang II binding was displaced by Ang II (K_i=7.6×10⁻⁹) and losartan (K_i=1.4×10⁻⁷) but also by the AT₂ receptor ligand PD 123319 (K_i=5.0×10⁻⁷). In addition, a low amount of AT₂ receptor binding was detected in the paraventricular nucleus using [¹²⁵I]CGP 42112 as radioligand, and the binding was displaced by Ang II (K_i=2.4×10⁻⁹), CGP 42112 (K_i=7.9×10⁻¹⁰), and PD 123319 (K_i=2.2×10⁻⁷). ACE, Ang II, and AT₁ as well as AT₂ receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.     

Forebrain neurogenesis after focal Ischemic and traumatic brain injury

Neural stem cells persist in the adult mammalian forebrain and are a potential source of neurons for repair after brain injury. The two main areas of persistent neurogenesis, the subventricular zone (SVZ)-olfactory bulb pathway and hippocampal dentate gyrus, are stimulated by brain insults such as stroke or trauma. Here we focus on the effects of focal cerebral ischemia on SVZ neural progenitor cells in experimental stroke, and the influence of mechanical injury on adult hippocampal neurogenesis in models of traumatic brain injury (TBI). Stroke potently stimulates forebrain SVZ cell proliferation and neurogenesis. SVZ neuroblasts are induced to migrate to the injured striatum, and to a lesser extent to the peri-infarct cortex. Controversy exists as to the types of neurons that are generated in the injured striatum, and whether adult-born neurons contribute to functional restoration remains uncertain. Advances in understanding the regulation of SVZ neurogenesis in general, and stroke-induced neurogenesis in particular, may lead to improved integration and survival of adult-born neurons at sites of injury. Dentate gyrus cell proliferation and neurogenesis similarly increase after experimental TBI. However, pre-existing neuroblasts in the dentate gyrus are vulnerable to traumatic insults, which appear to stimulate neural stem cells in the SGZ to proliferate and replace them, leading to increased numbers of new granule cells. Interventions that stimulate hippocampal neurogenesis appear to improve cognitive recovery after experimental TBI. Transgenic methods to conditionally label or ablate neural stem cells are beginning to further address critical questions regarding underlying mechanisms and functional significance of neurogenesis after stroke or TBI. Future therapies should be aimed at directing appropriate neuronal replacement after ischemic or traumatic injury while suppressing aberrant integration that may contribute to co-morbidities such as epilepsy or cognitive impairment.     

Selective CDK inhibitor limits neuroinflammation and progressive neurodegeneration after brain trauma

Traumatic brain injury (TBI) induces secondary injury mechanisms, including cell-cycle activation (CCA), which lead to neuronal cell death, microglial activation, and neurologic dysfunction. Here, we show progressive neurodegeneration associated with microglial activation after TBI induced by controlled cortical impact (CCI), and also show that delayed treatment with the selective cyclin-dependent kinase inhibitor roscovitine attenuates posttraumatic neurodegeneration and neuroinflammation. CCI resulted in increased cyclin A and D1 expressions and fodrin cleavage in the injured cortex at 6 hours after injury and significant neurodegeneration by 24 hours after injury. Progressive neuronal loss occurred in the injured hippocampus through 21 days after injury and correlated with a decline in cognitive function. Microglial activation associated with a reactive microglial phenotype peaked at 7 days after injury with sustained increases at 21 days. Central administration of roscovitine at 3 hours after CCI reduced subsequent cyclin A and D1 expressions and fodrin cleavage, improved functional recovery, decreased lesion volume, and attenuated hippocampal and cortical neuronal cell loss and cortical microglial activation. Furthermore, delayed systemic administration of roscovitine improved motor recovery and attenuated microglial activation after CCI. These findings suggest that CCA contributes to progressive neurodegeneration and related neurologic dysfunction after TBI, likely in part related to its induction of microglial activation.     

Cathepsin B contributes to traumatic brain injury‐induced cell death through a mitochondria‐mediated apoptotic pathway

It has been reported that lysosomal proteases play important roles in ischemic and excitotoxic neuronal cell death. We have previously reported that cathepsin B expression increased remarkably after traumatic brain injury (TBI). The present study sought to investigate the effects of a selective cathepsin B inhibitor (CBI) [N‐L‐3‐trans‐prolcarbamoyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline] on cell death and behavioral deficits in our model. We examined the levels of cathepsin B enzymatic activity and its expression by double labelling damaged cells in the brain slice with propidium iodide (PI) and anticathepsin B. The results showed an elevated enzymatic activity associated with TBI‐induced increase in a mature form of cathepsin B, suggesting that cathepsin B may play a role in TBI‐induced cell injury. PI was found to label cells positive for the neuronal‐specific nuclear marker NeuN, whereas fewer GFAP‐positive cells were labelled by PI, suggesting that neurons are more sensitive to cell death induced by TBI. Additionally, we found that pretreatment with CBI remarkably attenuated TBI‐induced cell death, lesion volume, and motor and cognitive dysfunction. To analyze the mechanism of action of cathepsin B in the cell death signaling pathway, we assessed DNA fragmentation by electrophoresis, Bcl‐2/Bax protein expression levels, Bid cleavage, cytochrome c release, and caspase‐3 activation. The results imply that cathepsin B contributes to TBI‐induced cell death through the present programmed cell necrosis and mitochondria‐mediated apoptotic pathways. © 2010 Wiley‐Liss, Inc.     

The Expression of FBP1 after Traumatic Brain Injury and Its Role in Astrocyte Proliferation

Far upstream element binding protein 1 (FBP1) has been identified as an upstream gene of p27^kip1 (p27), which is a key regulator of mammalian cell cycle regulation and neurogenesis. To elucidate the expression and function of FBP1 in central nervous system lesion and repair, we performed a traumatic brain injury (TBI) model in adult rats. We observed that FBP1 protein level significantly reduced at day 3 after injury, and the downregulation of FBP1 was predominant in astrocytes, which were largely proliferated after injury. Furthermore, in vitro, overexpression of FBP1 was concomitant with the up-regulation of p27 and reduction of PCNA in LPS-induced astrocyte proliferation. These results suggest that a decreased level of FBP1 in brain is involved in the proliferation of glial cells after TBI.     

Novel mGluR5 Positive Allosteric Modulator Improves Functional Recovery,Attenuates Neurodegeneration,and Alters Microglial Polarization after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.     

Age-dependent effect of apolipoprotein E4 on functional outcome after controlled cortical impact in mice

The apolipoprotein E4 (APOE4) gene leads to increased brain amyloid beta (Aβ) and poor outcome in adults with traumatic brain injury (TBI); however, its role in childhood TBI is controversial. We hypothesized that the transgenic expression of human APOE4 worsens the outcome after controlled cortical impact (CCI) in adult but not immature mice. Adult and immature APOE4 mice had worse motor outcome after CCI (P<0.001 versus wild type (WT)), but the Morris water maze performance was worse only in adult APOE4 mice (P=0.028 at 2 weeks, P=0.019 at 6 months versus WT), because immature APOE4 mice had performance similar to WT for up to 1 year after injury. Brain lesion size was similar in adult APOE4 mice but was decreased (P=0.029 versus WT) in injured immature APOE4 mice. Microgliosis was similar in all groups. Soluble brain Aβ₄₀ was increased at 48 hours after CCI in adult and immature APOE4 mice and in adult WT (P<0.05), and was dynamically regulated during the chronic period by APOE4 in adults but not immature mice. The data suggest age-dependent effects of APOE4 on cognitive outcome after TBI, and that therapies targeting APOE4 may be more effective in adults versus children with TBI.     

A New Avenue for Lithium: Intervention in Traumatic Brain Injury

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Neutralization of interleukin-1β modifies the inflammatory response and improves histological and cognitive outcome following traumatic brain injury in mice

Interleukin-1β (IL-1β) may play a central role in the inflammatory response following traumatic brain injury (TBI). We subjected 91 mice to controlled cortical impact (CCI) brain injury or sham injury. Beginning 5 min post-injury, the IL-1β neutralizing antibody IgG2a/k (1.5 μg/mL) or control antibody was infused at a rate of 0.25 μL/h into the contralateral ventricle for up to 14 days using osmotic minipumps. Neutrophil and T-cell infiltration and microglial activation was evaluated at days 1–7 post-injury. Cognition was assessed using Morris water maze, and motor function using rotarod and cylinder tests. Lesion volume and hemispheric tissue loss were evaluated at 18 days post-injury. Using this treatment strategy, cortical and hippocampal tissue levels of IgG2a/k reached 50 ng/mL, sufficient to effectively inhibit IL-1β in vitro . IL-1β neutralization attenuated the CCI-induced cortical and hippocampal microglial activation ( P  <   0.05 at post-injury days 3 and 7), and cortical infiltration of neutrophils ( P  <   0.05 at post-injury day 7). There was only a minimal cortical infiltration of activated T-cells, attenuated by IL-1β neutralization ( P  <   0.05 at post-injury day 7). CCI induced a significant deficit in neurological motor and cognitive function, and caused a loss of hemispheric tissue ( P  <   0.05). In brain-injured animals, IL-1β neutralizing treatment resulted in reduced lesion volume, hemispheric tissue loss and attenuated cognitive deficits ( P  <   0.05) without influencing neurological motor function. Our results indicate that IL-1β is a central component in the post-injury inflammatory response that, in view of the observed positive neuroprotective and cognitive effects, may be a suitable pharmacological target for the treatment of TBI.     

Distinct MRI pattern in lesional and perilesional area after traumatic brain injury in rat--11 months follow-up

To understand the dynamics of progressive brain damage after lateral fluid-percussion induced traumatic brain injury (TBI) in rat, which is the most widely used animal model of closed head TBI in humans, MRI follow-up of 11 months was performed. The evolution of tissue damage was quantified using MRI contrast parameters T₂, T_1ρ, diffusion (D_av), and tissue atrophy in the focal cortical lesion and adjacent areas: the perifocal and contralateral cortex, and the ipsilateral and contralateral hippocampus. In the primary cortical lesion area, which undergoes remarkable irreversible pathologic changes, MRI alterations start at 3 h post-injury and continue to progress for up to 6 months. In more mildly affected perifocal and hippocampal regions, the robust alterations in T₂, T_1ρ, and D_av at 3 h to 3 d post-injury normalize within the next 9-23 d, and thereafter, progressively increase for several weeks. The severity of damage in the perifocal and hippocampal areas 23 d post-injury appeared independent of the focal lesion volume. Magnetic resonance spectroscopy (MRS) performed at 5 and 10 months post-injury detected metabolic alterations in the ipsilateral hippocampus, suggesting ongoing neurodegeneration and inflammation. Our data show that TBI induced by lateral fluid-percussion injury triggers long-lasting alterations with region-dependent temporal profiles. Importantly, the temporal pattern in MRI parameters during the first 23 d post-injury can indicate the regions that will develop secondary damage. This information is valuable for targeting and timing interventions in studies aiming at alleviating or reversing the molecular and/or cellular cascades causing the delayed injury.     

N‐methyl‐D‐aspartate preconditioning improves short‐term motor deficits outcome after mild traumatic brain injury in mice

Traumatic brain injury (TBI) causes impairment of fine motor functions in humans and nonhuman mammals that often persists for months after the injury occurs. Neuroprotective strategies for prevention of the sequelae of TBI and understanding the molecular mechanisms and cellular pathways are related to the glutamatergic system. It has been suggested that cellular damage subsequent to TBI is mediated by the excitatory neurotransmitters, glutamate and aspartate, through the excessive activation of the N‐methyl‐D‐aspartate (NMDA) receptors. Thus, preconditioning with a low dose of NMDA was used as a strategy for protection against locomotor deficits observed after TBI in mice. Male adult mice CF‐1 were preconditioned with NMDA (75 mg/kg) 24 hr before the TBI induction. Under anesthesia with O₂/N₂O (33%: 66%) inhalation, the animals were subjected to the experimental model of trauma that occurs by the impact of a 25 g weight on the skull. Sensorimotor gating was evaluated at 1.5, 6, or 24 hr after TBI induction by using footprint and rotarod tests. Cellular damage also was assessed 24 hr after occurrence of cortical trauma. Mice preconditioned with NMDA were protected against all motor deficits revealed by footprint tests, but not those observed in rotarod tasks. Although mice showed motor deficits after TBI, no cellular damage was observed. These data corroborate the hypothesis that glutamatergic excitotoxicity, especially via NMDA receptors, contributes to severity of trauma. They also point to a putative neuroprotective mechanism induced by a sublethal dose of NMDA to improve motor behavioral deficits after TBI. © 2009 Wiley‐Liss, Inc.     

N-arachidonoyl-L-serine (AraS) possesses proneurogenic properties in vitro and in vivo after traumatic brain injury

N-arachidonoyl-L-serine (AraS) is a novel neuroprotective endocannabinoid. We aimed to test the effects of exogenous AraS on neurogenesis after traumatic brain injury (TBI). The effects of AraS on neural progenitor cells (NPC) proliferation, survival, and differentiation were examined in vitro. Next, mice underwent TBI and were treated with AraS or vehicle. Lesion volumes and clinical outcome were evaluated and the effects on neurogenesis were tested using immunohistochemistry. Treatment with AraS led to a dose-dependent increase in neurosphere size without affecting cell survival. These effects were partially reversed by CB1, CB2, or TRPV1 antagonists. AraS significantly reduced the differentiation of NPC in vitro to astrocytes or neurons and led to a 2.5-fold increase in expression of the NPC marker nestin. Similar effects were observed in vivo in mice treated with AraS 7 days after TBI. These effects were accompanied by a reduction in lesion volume and an improvement in neurobehavioral function compared with controls. AraS increases proliferation of NPCs in vitro in cannabinoid-receptor-mediated mechanisms and maintains NPC in an undifferentiated state in vitro and in vivo. Moreover, although given at 7 days post injury, these effects are associated with significant neuroprotective effects leading to an improvement in neurobehavioral functions.