A Novel Closed-Head Model of Mild Traumatic Brain Injury Using Focal Primary Overpressure Blast to the Cranium in Mice.

Mild traumatic brain injury (TBI) from focal head impact is the most common form of TBI in humans. Animal models, however, typically use direct impact to the exposed dura or skull, or blast to the entire head. We present a detailed characterization of a novel overpressure blast system to create focal closed-head mild TBI in mice. A high-pressure air pulse limited to a 7.5 mm diameter area on the left side of the head overlying the forebrain is delivered to anesthetized mice. The mouse eyes and ears are shielded, and its head and body are cushioned to minimize movement. This approach creates mild TBI by a pressure wave that acts on the brain, with minimal accompanying head acceleration-deceleration. A single 20-psi blast yields no functional deficits or brain injury, while a single 25-40 psi blast yields only slight motor deficits and brain damage. By contrast, a single 50-60 psi blast produces significant visual, motor, and neuropsychiatric impairments and axonal damage and microglial activation in major fiber tracts, but no contusive brain injury. This model thus reproduces the widespread axonal injury and functional impairments characteristic of closed-head mild TBI, without the complications of systemic or ocular blast effects or head acceleration that typically occur in other blast or impact models of closed-skull mild TBI. Accordingly, our model provides a simple way to examine the biomechanics, pathophysiology, and functional deficits that result from TBI and can serve as a reliable platform for testing therapies that reduce brain pathology and deficits.

A novel closed-head model of mild traumatic brain injury caused by primary overpressure blast to the cranium produces sustained emotional deficits in mice.

Emotional disorders are a common outcome from mild traumatic brain injury (TBI) in humans, but their pathophysiological basis is poorly understood. We have developed a mouse model of closed-head blast injury using an air pressure wave delivered to a small area on one side of the cranium, to create mild TBI. We found that 20-psi blasts in 3-month-old C57BL/6 male mice yielded no obvious behavioral or histological evidence of brain injury, while 25-40 psi blasts produced transient anxiety in an open field arena but little histological evidence of brain damage. By contrast, 50-60 psi blasts resulted in anxiety-like behavior in an open field arena that became more evident with time after blast. In additional behavioral tests conducted 2-8 weeks after blast, 50-60 psi mice also demonstrated increased acoustic startle, perseverance of learned fear, and enhanced contextual fear, as well as depression-like behavior and diminished prepulse inhibition. We found no evident cerebral pathology, but did observe scattered axonal degeneration in brain sections from 50 to 60 psi mice 3-8 weeks after blast. Thus, the TBI caused by single 50-60 psi blasts in mice exhibits the minimal neuronal loss coupled to "diffuse" axonal injury characteristic of human mild TBI. A reduction in the abundance of a subpopulation of excitatory projection neurons in basolateral amygdala enriched in Thy1 was, however, observed. The reported link of this neuronal population to fear suppression suggests their damage by mild TBI may contribute to the heightened anxiety and fearfulness observed after blast in our mice. Our overpressure air blast model of concussion in mice will enable further studies of the mechanisms underlying the diverse emotional deficits seen after mild TBI.

Motor, visual and emotional deficits in mice after closed-head mild traumatic brain injury are alleviated by the novel CB2 inverse agonist SMM-189.

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50-60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50-60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.

Mechanism of anti-glioma activity and in vivo efficacy of the cannabinoid ligand KM-233.

Glioblastoma multiforme (GBM) is the most common and devastating form of primary central nervous system malignancy. The prognosis for patients diagnosed with GBM is poor, having a median survival rate of 12-15 months. Despite modern advances in the development of antineoplastic agents, the efficacy of newer anti-cancer agents in the treatment of GBM is yet to be determined. Thus, there remains a significant unmet need for new therapeutic strategies against GBM. A promising chemotherapeutic intervention has emerged from studies of cannabinoid receptor agonists wherein tetrahydrocannabinol has been the most extensively studied. The novel cannabinoid ligand KM-233 was developed as a lead platform for future optimization of biopharmaceutical properties of classical based cannabinoid ligands. Treatment of U87MG human GBM cells with KM-233 caused a time dependent change in the phosphorylation profiles of MEK, ERK1/2, Akt, BAD, STAT3, and p70S6K. Almost complete mitochondrial depolarization was observed 6 h post-treatment followed by a rapid increase in cleaved caspase 3 and significant cytoskeletal contractions. Treatment with KM-233 also resulted in a redistribution of the Golgi-endoplasmic reticulum structures. Dose escalation studies in the orthotopic model using U87MG cells revealed an 80 % reduction in tumor size after 12 mg/kg daily dosing for 20 days. The evaluation of KM-233 against primary tumor tissue in the side flank model revealed a significant decrease in the rate of tumor growth. These findings indicate that structural refinement of KM-233 to improve its biopharmaceutical properties may lead to a novel and efficacious treatment for GBM.

Heterozygous deletion of NR1 subunit of the NMDA receptor alters ethanol-related behaviors and regional expression of NR2 subunits in the brain.

NMDA receptors have been hypothesized to play a role in various aspects of ethanol-related phenotypes, notably in ethanol withdrawal. However, the role of each of the specific subunits remains unclear. To address this issue, mice that are heterozygous for the NR1 deletion, and thus have a reduction in functional NMDA receptors, were examined for ethanol consumption and acute ethanol withdrawal. Additionally, mice were examined for the level of vocalization following footshock, and behavior in an elevated plus maze, to determine their responses to stress. In these behavioral tests, NR1 heterozygous mice were shown to consume significantly higher levels of ethanol in the two bottle-choice test showing a possible role for this receptor in ethanol consumption. Analysis of acute withdrawal found that the heterozygous mice exhibit lower levels of handling-induced convulsions consistent with a role in ethanol sensitivity or withdrawal. In contrast, no effects on stress-related phenotypes were detected. Levels of NR2A-NR2D subunits of the NMDA receptor in specific brain regions were compared between NR1+/- mice and wild-type controls to assess whether the behavioral responses were specific to the diminution in NR1 expression or whether these changes could be due to secondary changes in expression of other NMDA subunits. Real-time quantitative PCR, Western blot and immunohistochemistry were used to examine expression levels in the hippocampus, neocortex, striatum and cerebellum. For the majority of the subunits, no differences were found between the wild-type and heterozygous mice in any of the brain regions. However, the NR2B subunit exhibited differences in expression of RNA in the hippocampus and protein levels in multiple brain regions, between wild-type and NR1+/- mice. These results show that NR1 plays a role, through mechanisms as yet unknown, in the expression of NR2 subunits in a region and subtype specific manner. This provides evidence of the effects of altered levels of NR1 expression on ethanol withdrawal and consumption, and suggests that concomitant changes in the levels of NR2B may contribute to that effect.

Alcohol exposure during the first two trimesters-equivalent alters the development of corpus callosum projection neurons in the rat.

Children exposed prenatally to alcohol can display a variety of neural deficits, including an altered development of the corpus callosum (CC), the largest interhemispheric axon pathway in the brain. Furthermore, these children show functional abnormalities that are related to brain regions with significant numbers of CC connections. Little is known about how alcohol imparts influence on CC development, but one possible mechanism is by affecting the corpus callosum projection neurons (CCpn) directly. The purpose of this study was to quantify the effects of prenatal alcohol exposure on the number, size, and distribution of CCpn within the visual cortex. The visual cortex was selected specifically due to the many vision-related deficits noted in fetal alcohol exposed children and because the critical role of the CC in visual cortex development is well documented. Sprague-Dawley rat pups received one of four alcohol dosages during gestational days (G) 1-20, or reared as nutritional or untreated control animals. Each litter was categorized according to the peak blood alcohol concentration experienced. Pups were removed from each litter on days equivalent to G29, G36, G43, and G50, for histology and measurement. Callosal axons were labeled retrogradely to their CCpn using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and the CCpn were then examined using confocal laser scanning microscopy. Differences between alcohol-exposed and control animals were observed in CCpn cell body size, number, and location with the cortex. This was particularly true of animals exposed to high doses of alcohol. In addition, some trends of CCpn development were found to be unchanged as a result of prenatal alcohol exposure. The results demonstrate clear differences in the development of CCpn in the visual cortex between alcohol-exposed and control animals and suggest that this development is particularly affected in those animals exposed to high doses of alcohol.

Combined exposure to nicotine and ethanol throughout full gestation results in enhanced acquisition of nicotine self-administration in young adult rat offspring.

RATIONALE: Epidemiological evidence shows positive correlation between either maternal cigarette smoking or alcohol consumption on subsequent drug-taking behavior in offspring. However, the consequences of full gestational exposure to both drugs have not been studied experimentally despite concurrent use frequently reported among women of childbearing age. Such comorbid gestational drug exposure may increase susceptibility to acquiring cigarette smoking (i.e., nicotine self-administration), a major gateway drug. OBJECTIVES: We developed a noninvasive rat model for exposure to both nicotine (2-6 mg kg(-1) day(-1)) and EtOH (4 g/kg gavage) that continued throughout pregnancy and postnatal (P) days 2-12, the rodent equivalent of the human third trimester, a critical brain developmental period. Offspring with this full gestational exposure to both drugs (Nic+EtOH) were compared to controls: nicotine alone, EtOH alone, pair-fed (comparable nutrition and handling), and ad libitum chow-fed. At P60-90, offspring had unlimited chronic access to acquire i.v. nicotine self-administration. RESULTS: There were no differences in gender ratio, stillbirths, birth weights, righting reflex, eye opening age, or weight gain. However, Nic+EtOH offspring of both genders acquired nicotine self-administration (15 or 30 microg kg(-1) injection(-1)) more rapidly, at a higher percentage, and at a higher level than offspring in the other cohorts. CONCLUSION: Full gestational Nic+EtOH exposure produced no overt alterations in standard postnatal measures but resulted in an enhanced acquisition of nicotine self-administration in young adult offspring.

Prenatal alcohol exposure delays the development of the cortical barrel field in neonatal rats.

In-utero alcohol exposure produces sensorimotor developmental abnormalities that often persist into adulthood. The rodent cortical barrel field associated with the representation of the body surface was used as our model system to examine the effect of prenatal alcohol exposure (PAE) on early somatosensory cortical development. In this study, pregnant female rats were intragastrically gavaged daily with high doses of alcohol (6 gm/kg body weight) throughout the first 20 days of pregnancy. Blood alcohol levels were measured in the pregnant dams on gestational days 13 (G13) and G20. The ethanol treated group (EtOH) was compared to the normal control chowfed (CF) group, nutritionally matched pairfed (PF) group, and cross-foster (XF) group. Cortical barrel development was examined in pups across all treatment groups from G25, corresponding to postnatal day 2 (P2), to G32 corresponding to P9. The EtOH and control group pups were weighed, anesthetized, and perfused. Brains were removed and weighed with, and without cerebellum and olfactory bulbs, and neocortex was removed and weighed. Cortices were then flattened, sectioned tangentially, and stained with a metabolic marker, cytochrome oxidase (CO) to reveal the barrel field. Progression of barrel development was distinguished into three categories: (a) absent, (b) cloudy barrel-like pattern, and (c) well-formed barrels with intervening septae. The major findings are: (1) PAE delayed barrel field development by one or more days, (2) the barrel field first appeared as a cloudy pattern that gave way on subsequent days to an adult-like pattern with clearly demarcated intervening septal regions, (3) the barrel field developed differentially in a lateral-to-medial gradient in both alcohol and control groups, (4) PAE delayed birth by one or more days in 53% of the pups, (5) regardless of whether pups were born on G23 (normal expected birth date for non-alcohol controls) or as in the case for the alcohol-delayed pups born as late as G27, the barrel field was never present at birth suggesting the importance of postnatal experience on barrel field development, and (6) PAE did not disrupt the normal barrel field pattern, although both total body and brain weights were compromised. These findings suggest that PAE delays the development of the somatosensory cortex (SI); such delays may interfere with timing and formation of cortical circuits. It is unknown whether other nuclei along the somatosensory pathway undergo similar delays in development or if PAE selectively disrupts cortical circuitry.

Prenatal alcohol exposure (PAE) reduces the size of the forepaw representation in forepaw barrel subfield (FBS) cortex in neonatal rats: relationship between periphery and central representation.

Prenatal alcohol exposure (PAE) alters limb development that may lead to structural and functional abnormalities of the limb reported in children diagnosed with Fetal Alcohol Spectrum Disorder. To determine whether PAE alters the central representation of the forelimb we used the rodent barrel cortex as our model system where it was possible to visualize and quantitatively measure the size of the forepaw representation in the forepaw barrel subfield (FBS) in first somatosensory cortex. In the present study, we examined the effects of PAE on pattern and size of the forepaw and forepaw representation in FBS in neonatal rats at gestational day 32 that corresponds to postnatal day 9. Pregnant Sprague-Dawley rats were chronically intubated with binge doses of ethanol (6 g/kg) from gestational day 1 through gestational day 20. The offspring of the ethanol treated dams comprised the ethanol (EtOH) group. The effect of PAE on the EtOH group was compared with a nutritional-controlled pairfed (PF) group and a normal chowfed (CF) group. The ventral (glabrous) surface area of the forepaw digits, length of digit 2 through digit 5, and the corresponding glabrous forepaw digit representations in the FBS were measured and compared between treatment groups. In rats exposed to in utero alcohol, the sizes of the overall glabrous forepaw and forepaw digits were significantly reduced in EtOH pups compared to CF and PF pups; overall glabrous forepaw area was 11% smaller than CF controls. Glabrous digit lengths were also smaller in EtOH rats compared to CF controls and significantly smaller in digit 2 through digit 4. The glabrous digit representation in FBS was 18% smaller in the EtOH group when compared to the CF treatment. However, PAE did not produce malformations in the forepaw or alter the pattern of the forepaw representation in FBS; instead, PAE significantly reduced both body and brain weights compared to controls. Unexpectedly, little or no correlation was observed between the size of the glabrous forepaw compared to the size of the glabrous forepaw representation in the FBS for any of the treatment groups. The present findings of PAE-related alterations in sensory periphery and the central cortical representation may underlie deficits in sensorimotor integration reported among children with Fetal Alcohol Spectrum Disorder.

Prenatal alcohol exposure alters the size, but not the pattern, of the whisker representation in neonatal rat barrel cortex.

Maternal alcohol exposure results in a variety of neurodevelopmental abnormalities that include cognitive and sensorimotor dysfunctions that often persist into adulthood. Many reports of central nervous system disturbances associated within a clinical diagnosis of fetal alcohol syndrome point toward disturbances in central information processing. In this study, we used the rat barrel field cortex as a model system to examine the effects of prenatal alcohol exposure (PAE) on the organization and size of the large whisker representation in layer IV of the posteromedial barrel subfield (PMBSF) in somatosensory cortex. Pregnant rats (Sprague-Dawley) were intragastrically gavaged daily with alcohol doses (6 gm/kg body weight) from gestational day 1 to day 20 in a chronic binge pattern which produced blood alcohol levels ranging between 260 mg/dl and 324 mg/dl. Chow-fed (CF), pair-fed (PF), and cross-foster (XF) groups served as normal, nutritionally matched, and maternal controls, respectively, for the ethanol-exposed (EtOH) treatment group. All pups were examined on gestational day 32 corresponding approximately to postnatal day 9. EtOH and control group pups were weighed, anesthetized, and perfused. Brains were removed and weighed, with and without cerebellum and olfactory bulbs, and the neocortex was removed and weighed. Cortices were then flattened, sectioned tangentially, and stained with a metabolic marker-cytochrome oxidase-to reveal the barrel field. A subset of 27 cortical barrels, associated with the representation of the large whisker pad, was selected to examine in detail. The major results were: (i) the total barrel field area comprising the PMBSF was significantly reduced in EtOH (by 17%) and XF (by 16%) pups compared with CF pups, (ii) the sizes of individual barrels within the PMBSF were also significantly reduced in EtOH (16%) and XF (18%) pups, (iii) the septal region between barrels was also significantly reduced in EtOH (18%) and XF (12%) pups, (iv) anteriorly located barrels underwent greater reduction in size relative to the posteriorly located barrels, (v) body weights were also significantly reduced in EtOH (21%) and XF (27%) pups, (vi) total brain weight [with and without (forebrain) cerebellum/olfactory bulbs] and cortical weights were also significantly reduced in EtOH (total brain weight 15%, forebrain weight 16%, cortical weight 15%) and XF (18%, 19%, 20%) pups, and in contrast (vi) neither the overall barrel field pattern nor the pattern of individual barrels in the PMBSF was altered. These findings suggest that PAE reduces body and brain weight as well as the central cortical representation of the whisker pad, while leaving the overall barrel field pattern unperturbed. While these results might appear to support a miniaturization hypothesis (smaller PMBSF, smaller brain, smaller body weight), PAE also shows regional vulnerability within the PMBSF whereby anteriorly located barrels are most affected.

Corticothalamic and thalamocortical pathfinding in the mouse: dependence on intermediate targets and guidance axis.

Recently, increasing attention has been paid to the study of intermediate targets and their relay guidance role in long-range pathfinding. In the present study, mechanisms of corticothalamic and thalamocortical pathfinding were investigated in C57BL/6 mice using in vitro DiI labeling and in vivo cholera toxin labeling. Specifically, three important intermediate targets, the subplate, ganglionic eminence, and reticular thalamic nucleus, were studied for their role in corticothalamic and thalamocortical pathfinding. The results show that the neuroepithelium of the ganglionic eminence is a source of pioneer neurons and pioneer fibers. Through radial and tangential migration, these pioneer neurons and fibers can approach the differentiating field of the ganglionic eminence, the subplate and thalamic reticular nucleus to participate in the formation of these three intermediate targets. Furthermore, the subplate, ganglionic eminence and thalamic reticular nucleus are linked by pioneer neurons and fibers to form a guidance axis. The guidance axis and the three important intermediate targets provide an ideal environment of contact guidance and chemical guidance for the corticothalamic and thalamocortical pathfinding. The concept of a "waiting time" in the subplate and the thalamic reticular nucleus is likely due to the expression of a guidance effect, so that the thalamocortical and corticothalamic projections can be deployed spatially and temporally to the subplate and thalamic reticular nucleus before these projections enter their final destinations, the neocortex and thalamus.

Corpus callosum and visual cortex of mice with deletion of the NMDA-NR1 receptor: I. Accelerated development of callosal projection neurons.

Many pharmacological experiments show that the ionotropic receptor NMDA has both neurotrophic and neuroexcitotoxic effects. The neurotrophic function is manifested in many ways including acceleration of neuronal development, enhancement of neuronal migration, neuroprotection, blockage of apoptosis, prevention of aging and prematurity, as well as effects on synaptic plasticity and synaptogenesis. On the other hand, the neuroexcitotoxic function is manifested in its role in neurological and psychiatric diseases such as epilepsy, Parkinson's disease and schizophrenia. The present study explores the consequences of complete and partial absence of NMDA-NR1 receptors throughout development. Using DiI tracing in vitro, the development of corpus callosum projection neurons in transgenic mice with deletion of the NMDA-NR1 receptor was observed in visual cortex. Compared to littermate controls, the histogenesis and neuronal development of corpus callosum cells of origin was found to be accelerated in NR1-/- mice. That is, the corpus callosum projection neurons in NR1 knockout mice developed earlier and faster than in littermate heterozygous and wild-type mice. However, the corpus callosum projection neurons in NR1 heterozygous mice developed earlier and faster than in littermate wild-type mice. This suggests that NMDA-NR1 receptors are involved in sequencing and/or temporal regulation of neuronal development, and that there is a gene-dose effect. Studies from other laboratories suggest that the observed phenomenon of prematurity or accelerated development is a direct effect of altered expression of genes found in mice with deletion of the NMDA-NR1 receptor.

Corpus callosum and visual cortex of mice with deletion of the NMDA-NR1 receptor. II. Attenuation of prenatal alcohol exposure effects.

Offspring of transgenic mice with deletion of the NMDA-NR1 (NR1) receptor received prenatal alcohol exposure during most of gestation. Before and after birth, offspring were sacrificed in order to examine the morphological consequences of the prenatal exposure. Previously, we reported that the dendritic arborization of corpus callosum projection neurons (CCpn) in visual cortex was abnormal in rats given prenatal alcohol exposure; the effects were dose-dependent [Neurotoxicol. Teratol. 24 (2002) 719-732]. The same parameters were examined in the transgenic mice. Crystals of DiI were placed into the CC of mice at different ages that had been prenatally exposed to alcohol. Controls included untreated transgenic mice, and transgenic mice with the same nutritional and handling stressors. Compared to Controls, prenatal alcohol exposure caused the NR1+/+ mice to expand the dendritic arbor of CCpn in visual cortex. The dendritic arbors had increased branch numbers and length; these increases were dose-dependent. In contrast, the prenatally exposed NR1-/- mice showed normal dendritic arbors with all prenatal alcohol doses. In addition, prenatal alcohol exposure was found to have morbidity and teratogenic effects on offspring. In seven separate indicators of the effects of prenatal alcohol exposure, only one indicator was present but reduced in NR1-/- offspring, indicating that total deletion of the NMDA-NR1 receptor throughout development largely blocks but sometimes attenuates the effects of prenatal alcohol exposure. Similarly, in seven separate indicators of the effects of prenatal alcohol exposure, five indicators were attenuated in NR1+/- compared to NR1+/+ offspring, although affected more than in NR1-/-; this suggests a gene-dose effect. The results indicate that functional NMDA-NR1 receptors are necessary for the neurotoxic and teratogenic effects of prenatal alcohol exposure. This study will aid in understanding how the NMDA receptors play an important role in prenatal alcohol effects on brain development.

Second trimester prenatal alcohol exposure alters development of rat corpus callosum.

Prenatal alcohol exposure produces many developmental defects of the central nervous system (CNS), such as in the corpus callosum (CC). This study was designed to observe the effect of prenatal alcohol exposure during the second trimester equivalent on the development of dendritic arbors of CC projection neurons (CCpn) in rat visual cortex. In addition, the effect of second trimester equivalent prenatal alcohol exposure on brain weight was determined. Pregnant dams received 1.2-6.0 g/kg ethanol (EtOH) during gestational day (G) 11-20. Controls consisted of normal and nutritionally matched pairfed (PF) dams. Pups were sacrificed on the day of birth, G26, G29 and G33. DiI crystals were placed in the midsagittal CC bundle to retrogradely label CCpn. Images of visual cortex were obtained from tissue slices using a confocal laser scanning microscope. The number and length of apical and basilar dendrite branches were determined. The results show that prenatal alcohol exposure restricted to the second trimester equivalent alters the development of the CCpn dendritic arbor and the brain weight in a blood alcohol concentration (BAC)-dependent manner. The alteration in the EtOH CCpn is manifested as an increase in the number and length of CCpn apical and basilar dendrite branches, while brain weight is reduced compared with Controls.