Methamphetamine augment HIV-1 Tat mediated memory deficits by altering the expression of synaptic proteins and neurotrophic factors.

Methamphetamine (METH) abuse is common among individuals infected with HIV-1 and has been shown to affect HIV replication and pathogenesis. These HIV-1 infected individuals also exhibit greater neuronal injury and higher cognitive decline. HIV-1 proteins, specifically gp120 and HIV-1 Tat, have been earlier shown to affect neurocognition. HIV-1 Tat, a viral protein released early during HIV-1 replication, contributes to HIV-associated neurotoxicity through various mechanisms including production of pro-inflammatory cytokines, reactive oxygen species and dysregulation of neuroplasticity. However, the combined effect of METH and HIV-1 Tat on neurocognition and its potential effect on neuroplasticity mechanisms remains largely unknown. Therefore, the present study was undertaken to investigate the combined effect of METH and HIV-1 Tat on behavior and on the expression of neuroplasticity markers by utilizing Doxycycline (DOX)-inducible HIV-1 Tat (1-86) transgenic mice. Expression of Tat in various brain regions of these mice was confirmed by RT-PCR. The mice were administered with an escalating dose of METH (0.1 mg/kg to 6 mg/kg, i.p) over a 7-day period, followed by 6 mg/kg, i.p METH twice a day for four weeks. After three weeks of METH administration, Y maze and Morris water maze assays were performed to determine the effect of Tat and METH on working and spatial memory, respectively. Compared with controls, working memory was significantly decreased in Tat mice that were administered METH. Moreover, significant deficits in spatial memory were also observed in Tat-Tg mice that were administered METH. A significant reduction in the protein expressions of synapsin 1, synaptophysin, Arg3.1, PSD-95, and BDNF in different brain regions were also observed. Expression levels of Calmodulin kinase II (CaMKII), a marker of synaptodendritic integrity, were also significantly decreased in HIV-1 Tat mice that were treated with METH. Together, this data suggests that METH enhances HIV-1 Tat-induced memory deficits by reducing the expression of pre- and postsynaptic proteins and neuroplasticity markers, thus providing novel insights into the molecular mechanisms behind neurocognitive impairments in HIV-infected amphetamine users.

Exercise preconditioning improves traumatic brain injury outcomes.

PURPOSE: To determine whether 6 weeks of exercise performed prior to traumatic brain injury (TBI) could improve post-TBI behavioral outcomes in mice, and if exercise increases neuroprotective molecules (vascular endothelial growth factor-A [VEGF-A], erythropoietin [EPO], and heme oxygenase-1 [HO-1]) in brain regions responsible for movement (sensorimotor cortex) and memory (hippocampus). METHODS: 120 mice were randomly assigned to one of four groups: (1) no exercise+no TBI (NOEX-NOTBI [n=30]), (2) no exercise+TBI (NOEX-TBI [n=30]), (3) exercise+no TBI (EX-NOTBI [n=30]), and (4) exercise+TBI (EX-TBI [n=30]). The gridwalk task and radial arm water maze were used to evaluate sensorimotor and cognitive function, respectively. Quantitative real time polymerase chain reaction and immunostaining were performed to investigate VEGF-A, EPO, and HO-1 mRNA and protein expression in the right cerebral cortex and ipsilateral hippocampus. RESULTS: EX-TBI mice displayed reduced post-TBI sensorimotor and cognitive deficits when compared to NOEX-TBI mice. EX-NOTBI and EX-TBI mice showed elevated VEGF-A and EPO mRNA in the cortex and hippocampus, and increased VEGF-A and EPO staining of sensorimotor cortex neurons 1 day post-TBI and/or post-exercise. EX-TBI mice also exhibited increased VEGF-A staining of hippocampal neurons 1 day post-TBI/post-exercise. NOEX-TBI mice demonstrated increased HO-1 mRNA in the cortex (3 days post-TBI) and hippocampus (3 and 7 days post-TBI), but HO-1 was not increased in mice that exercised. CONCLUSIONS: Improved TBI outcomes following exercise preconditioning are associated with increased expression of specific neuroprotective genes and proteins (VEGF-A and EPO, but not HO-1) in the brain.

Behavioral effects and mechanisms of migraine pathogenesis following estradiol exposure in a multibehavioral model of migraine in rat.

Migraine is one of the most common neurological disorders, leading to more than 1% of total disability reported and over 68 million visits to emergency rooms or physician's offices each year in the United States. Three times as many women as men have migraine, and while the mechanism behind this is not well understood, 17ß-estradiol (estradiol) has been implicated to play a role. Studies have demonstrated that exposure to estrogen can lead to activation of inflammatory pathways, changes in sodium gated channel activity, as well as enhanced vasodilation and allodynia. Estradiol receptors are found in trigeminal nociceptors, which are involved in signaling during a migraine attack. The purpose of this study was to investigate the role of estradiol in migraine pathogenesis utilizing a multibehavioral model of migraine in rat. Animals were surgically implanted with a cannula system to induce migraine and behavior was assessed following exposure to a proestrus level of estradiol for total locomotor activity, light and noise sensitivity, evoked grooming patterns, and enhanced acoustic startle response. Results demonstrated decreased locomotor activity, increased light and noise sensitivity, altered facial grooming indicative of allodynia and enhanced acoustic startle. Further examination of tissue samples revealed increased expression of genes associated with inflammation and vasodilation. Overall, this study demonstrates exacerbation of migraine-like behaviors following exposure to estradiol and helps further explain the underlying mechanisms behind sex differences found in this common neurological disorder.

Differential response of miRNA-21 and its targets after traumatic brain injury in aging mice.

The present study investigated the possible role of miR-21, a miRNA that has known prosurvival function, in poor outcomes in the elderly following traumatic brain injury compared to adults. Controlled cortical impact injury was induced in adult (5-6 months) and aged (22-24 months) C57/BL6 mice. miR-21 and four of its targets (PDCD4, TIMP3, RECK, PTEN) were analyzed at 1, 3, 7 days post injury in samples of injured cortex using real-time PCR analysis. Basal miR-21 expression was higher in the aged brain than in the adult brain. In the adult brain, miR-21 expression increased in response to injury, with the maximum increase 24 hours after injury followed by a gradual decrease, returning to baseline 7 days post-injury. In contrast, in aged mice, miR21 showed no injury response, and expression of miR-21 target genes (PTEN, PDCD4, RECK, TIMP3) was up-regulated at all post injury time points, with a maximal increase at 24 hours post injury. Based on these results, we conclude that the diminished miR21 injury response in the aged brain leads to up-regulation of its targets, with the potential to contribute to the poor prognosis following TBI in aging brain. Therefore, strategies aimed at up-regulation of miR-21 and/or down regulation of its targets might be useful in improving outcomes in the elderly following TBI.

Neuroglobin overexpression improves sensorimotor outcomes in a mouse model of traumatic brain injury.

There is a significant need for novel treatments that will improve traumatic brain injury (TBI) outcomes. One potential neuroprotective mechanism is to increase oxygen binding proteins such as neuroglobin. Neuroglobin has a high affinity for oxygen, is an effective free radical scavenger, and is neuroprotective within the brain following hypoxia and ischemia. The purpose of this study was to determine whether neuroglobin overexpression improves sensorimotor outcomes following TBI in transgenic neuroglobin overexpressing (NGB) mice. Additional study aims were to determine if and when an endogenous neuroglobin response occurred following TBI in wild-type (WT) mice, and in what brain regions and cell types the response occurred. Controlled cortical impact (CCI) was performed in adult (5 month) C57/BL6 WT mice, and NGB mice constitutively overexpressing neuroglobin via the chicken beta actin promoter coupled with the cytomegalovirus distal enhancer. The gridwalk task was used for sensorimotor testing of both WT and NGB mice, prior to injury, and at 2, 3, and 7 days post-TBI. NGB mice displayed significant reductions in the average number of foot faults per minute walking at 2, 3, and 7 days post-TBI when compared to WT mice at each time point. Neuroglobin mRNA expression was assessed in the injured cortex of WT mice prior to injury, and at 1, 3, 7, and 14 days post-TBI using quantitative real time polymerase chain reaction (qRT-PCR). Neuroglobin mRNA was significantly increased at 7 days post-TBI. Immunostaining showed neuroglobin primarily localized to neurons and glial cells in the injured cortex and ipsilateral hippocampus of WT mice, while neuroglobin was present in all brain regions of NGB mice at 7 days post-TBI. These results showed that overexpression of neuroglobin reduced sensorimotor deficits following TBI, and that an endogenous increase in neuroglobin expression occurs during the subacute period. Increasing neuroglobin expression through novel therapeutic interventions during the acute period after TBI may improve recovery.

Exposure to bisphenol A exacerbates migraine-like behaviors in a multibehavior model of rat migraine.

Migraine is a common and debilitating neurological disorder suffered worldwide. Women experience this condition 3 times more frequently than men, with estrogen strongly implicated to play a role. Bisphenol A (BPA), a highly prevalent xenoestrogen, is known to have estrogenic activity and may have an effect in migraine onset, intensity, and duration through estrogen receptor signaling. It was hypothesized that BPA exposure exacerbates migraine symptoms through estrogen signaling and downstream activation of nociception related pathways. Utilizing a multibehavior model of migraine in ovariectomized female rats, changes in locomotion, light and sound sensitivity, grooming, and acoustic startle were examined. Furthermore, changes in the expression of genes related to estrogen (ERα, GPR30), and nociception (extracellular signal regulated kinase, ERK, sodium gated channel, Nav1.8, and fatty acid amide hydrolase, FAAH) were studied following behavioral experiments. The following results were obtained: BPA treatment significantly exacerbated migraine-like behaviors in rats. Rats exposed to BPA demonstrated decreased locomotion, exacerbated light and sound aversion, altered grooming habits, and enhanced startle reflexes. Furthermore, BPA exposure increased mRNA expression of estrogen receptors, total ERK mRNA and ERK activation, as well as Nav1.8, and FAAH mRNA, indicative of altered estrogen signaling and altered nociception. These results show that BPA, an environmentally pervasive xenoestrogen, exacerbates migraine-like behavior in a rat model and alters expression of estrogen and nociception-related genes.

Low brain DHA content worsens sensorimotor outcomes after TBI and decreases TBI-induced Timp1 expression in juvenile rats.

The effects of dietary modulation of brain DHA content on outcomes after TBI were examined in a juvenile rat model. Long-Evans rats with normal or diet-induced decreases in brain DHA were subjected to a controlled cortical impact or sham surgery on postnatal day 17. Rats with the greatest decreases in brain DHA had the poorest sensorimotor outcomes after TBI. Ccl2, Gfap, and Mmp 9 mRNA levels, and MMP-2 and -9 enzymatic activities were increased after TBI regardless of brain DHA level. Lesion volume was not affected by brain DHA level. In contrast, TBI-induced Timp1 expression was lower in rats on the Deficient diet and correlated with brain DHA level. These data suggest that decreased brain DHA content contributes to poorer sensorimotor outcomes after TBI through a mechanism involving modulation of Timp1 expression.

Pathogenic implications of iron accumulation in multiple sclerosis.

Iron, an essential element used for a multitude of biochemical reactions, abnormally accumulates in the CNS of patients with multiple sclerosis (MS). The mechanisms of abnormal iron deposition in MS are not fully understood, nor do we know whether these deposits have adverse consequences, that is, contribute to pathogenesis. With some exceptions, excess levels of iron are represented concomitantly in multiple deep gray matter structures often with bilateral representation, whereas in white matter, pathological iron deposits are usually located at sites of inflammation that are associated with veins. These distinct spatial patterns suggest disparate mechanisms of iron accumulation between these regions. Iron has been postulated to promote disease activity in MS by various means: (i) iron can amplify the activated state of microglia resulting in the increased production of proinflammatory mediators; (ii) excess intracellular iron deposits could promote mitochondria dysfunction; and (iii) improperly managed iron could catalyze the production of damaging reactive oxygen species (ROS). The pathological consequences of abnormal iron deposits may be dependent on the affected brain region and/or accumulation process. Here, we review putative mechanisms of enhanced iron uptake in MS and address the likely roles of iron in the pathogenesis of this disease.

Effects of aging on blood brain barrier and matrix metalloproteases following controlled cortical impact in mice.

Aging alters the ability of the brain to respond to injury. One of the major differences between the adult and aged brain is that comparable injuries lead to greater blood brain barrier disruption in the aged brain. The goals of these studies were to quantify the effects of age on BBB permeability using high field strength MRI T1 mapping and to determine whether activation of matrix metalloproteases, their inhibitors, or expression of blood brain barrier structural proteins, occludin, zonnula occludins-1 (ZO-1) and claudin-5 were altered following injury to the aged C57/BL6 mouse brain. T1 mapping studies revealed greater blood brain barrier permeability in the aged (21-24 months old) brain than in the adult (4-6 months old) following controlled cortical impact. The increased blood brain barrier permeability in the pericontusional region was confirmed with IgG immunohistochemistry. MMP-9 activity was increased following controlled cortical impact in the aged brain, and this was accompanied by increased MMP-9 gene expression. MMP-2 activity was higher in the uninjured aged brain than in the adult brain. Occludin and ZO-1 mRNA levels were unchanged following injury in either age group, but claudin-5 mRNA levels were lower in the aged than the adult brain following injury. These results demonstrate quantitative increases in blood brain barrier permeability in the aged brain following injury that are accompanied by increased MMP-9 activation and decreased blood brain barrier repair responses.

Iron deposition is independent of cellular inflammation in a cerebral model of multiple sclerosis.

BACKGROUND: Perivenular inflammation is a common early pathological feature in multiple sclerosis (MS). A recent hypothesis stated that CNS inflammation is induced by perivenular iron deposits that occur in response to altered blood flow in MS subjects. In order to evaluate this hypothesis, an animal model was developed, called cerebral experimental autoimmune encephalomyelitis (cEAE), which presents with CNS perivascular iron deposits. This model was used to investigate the relationship of iron deposition to inflammation. METHODS: In order to generate cEAE, mice were given an encephalitogen injection followed by a stereotactic intracerebral injection of TNF-α and IFN-γ. Control animals received encephalitogen followed by an intracerebral injection of saline, or no encephalitogen plus an intracerebral injection of saline or cytokines. Laser Doppler was used to measure cerebral blood flow. MRI and iron histochemistry were used to localize iron deposits. Additional histological procedures were used to localize inflammatory cell infiltrates, microgliosis and astrogliosis. RESULTS: Doppler analysis revealed that cEAE mice had a reduction in cerebral blood flow compared to controls. MRI revealed T2 hypointense areas in cEAE animals that spatially correlated with iron deposition around vessels and at some sites of inflammation as detected by iron histochemistry. Vessels with associated iron deposits were distributed across both hemispheres. Mice with cEAE had more iron-labeled vessels compared to controls, but these vessels were not commonly associated with inflammatory cell infiltrates. Some iron-laden vessels had associated microgliosis that was above the background microglial response, and iron deposits were observed within reactive microglia. Vessels with associated astrogliosis were more commonly observed without colocalization of iron deposits. CONCLUSION: The findings indicate that iron deposition around vessels can occur independently of inflammation providing evidence against the hypothesis that iron deposits account for inflammatory cell infiltrates observed in MS.

Sex-related differences in animal models of migraine headache.

Trigeminal nerve-mediated pain disorders such as migraine, temporomandibular joint disorder, and classical trigeminal neuralgia are more prevalent in women than in men. Female laboratory animals also show greater responses to various nociceptive stimuli than male animals. However, current knowledge of migraine pathogenesis is based primarily on experimental studies conducted in male animals and lack of migraine research with female animals limits clinical relevance. Migraine is triggered by any alteration in the intrinsic or extrinsic milieu and women at reproductive age are continuously prone to waxing and waning effects of female sex hormones. The experimental approach to this problem is complex because the rodent estrous cycle differs from the human cycle, and because exogenous hormone replacement in ovariectomized females has its limitations. The existence of multiple estrogen receptors in the trigeminal system also presents a challenge. Estrogens do not seem to directly affect calcitonin gene-related peptide or 5-HT(1D) receptors in the trigeminal system. Nonetheless, 2 estrogen receptors activate MAPK/ERK signaling pathway that mediates nociceptive processing in trigeminal nucleus caudalis. In addition, estrogen enhances susceptibility to cortical spreading depression, the neurobiological event underlying migraine aura, which may be independent of the estrous cycle. Further studies in female animals are required to clarify mechanisms underlying sex differences with respect to fluctuating sex hormones, cortical spreading depression, and excitability of the trigeminovascular system.

Mechanisms of pain modulation by sex hormones in migraine.

A number of pain conditions, acute as well as chronic, are much more prevalent in women, such as temporomandibular disorder (TMD), irritable bowel syndrome, fibromyalgia, and migraine. The association of female sex steroids with these nociceptive conditions is well known, but the mechanisms of their effects on pain signaling are yet to be deciphered. We reviewed the mechanisms through which female sex steroids might influence the trigeminal nociceptive pathways with a focus on migraine. Sex steroid receptors are located in trigeminal circuits, providing the molecular substrate for direct effects. In addition to classical genomic effects, sex steroids exert rapid nongenomic actions to modulate nociceptive signaling. Although there are only a handful of studies that have directly addressed the effect of sex hormones in animal models of migraine, the putative mechanisms can be extrapolated from observations in animal models of other trigeminal pain disorders, like TMD. Sex hormones may regulate sensitization of trigeminal neurons by modulating expression of nociceptive mediator such as calcitonin gene-related peptide. Its expression is mostly positively regulated by estrogen, although a few studies also report an inverse relationship. Serotonin (5-Hydroxytryptamine [5-HT]) is a neurotransmitter implicated in migraine; its synthesis is enhanced in most parts of brain by estrogen, which increases expression of the rate-limiting enzyme tryptophan hydroxylase and decreases expression of the serotonin re-uptake transporter. Downstream signaling, including extracellular signal-regulated kinase activation, calcium-dependent mechanisms, and cAMP response element-binding activation, are thought to be the major signaling events affected by sex hormones. These findings need to be confirmed in migraine-specific animal models that may also provide clues to additional ion channels, neuropeptides, and intracellular signaling cascades that contribute to the increased prevalence of migraine in women.

Sensorimotor behavioral tests for use in a juvenile rat model of traumatic brain injury: assessment of sex differences.

Modeling juvenile traumatic brain injury (TBI) in rodents presents several unique challenges compared to adult TBI, one of which is selecting appropriate sensorimotor behavioral tasks that enable the assessment of the extent of injury and recovery over time in developing animals. To address this challenge, we performed a comparison of common sensorimotor tests in Long-Evans rats of various sizes and developmental stages (postnatal days 16-45, 35-190 g). Tests were compared and selected for their developmental appropriateness, scalability for growth, pre-training requirements, and throughput capability. Sex differences in response to TBI were also assessed. Grid walk, automated gait analysis, rotarod, beam walk, spontaneous forelimb elevation test, and measurement of motor activity using the force-plate actometer were evaluated. Grid walk, gait analysis, and rotarod failed to meet one or more of the evaluation criteria. Beam walk, spontaneous forelimb elevation test, and measurement of motor activity using the force-plate actometer satisfied all criteria and were capable of detecting motor abnormalities in rats subjected to controlled cortical impact on postnatal day 17. No sex differences were detected in the acute effects of TBI or functional recovery during the 28 days after injury using these tests. This demonstrates the utility of these tests for the evaluation of sensorimotor function in studies using rat models of pediatric TBI, and suggests that pre-pubertal males and females respond similarly to TBI with respect to sensorimotor outcomes.

Sex differences in behavior and expression of CGRP-related genes in a rodent model of chronic migraine.

OBJECTIVE: The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene-related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. BACKGROUND: Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate-buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura. METHODS: Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP. RESULTS: Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP-related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP-related genes were upregulated by both IS and PBS applications. CONCLUSIONS: This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation.

Upregulation of inflammatory mediators in a model of chronic pain after spinal cord injury.

Chronic neuropathic pain is a disabling condition observed in large number of individuals following spinal cord injury (SCI). Recent progress points to an important role of neuroinflammation in the pathogenesis of central neuropathic pain. The focus of the present study is to investigate the role of proinflammatory molecules IL-1ß, TNF-α, MCP-1, MMP-9 and TIMP-1 in chronic neuropathic pain in a rodent model of SCI. Rats were subjected to spinal cord contusion using a controlled linear motor device with an injury epicenter at T10. The SCI rats had severe impairment in locomotor function at 7 days post-injury as assessed by the BBB score. The locomotor scores showed significant improvement starting at day 14 and thereafter showed no further improvement. The Hargreaves' test was used to assess thermal hyperalgesia for hindpaw, forepaw and tail. A significant reduction in withdrawal latency was observed for forepaw and tail of SCI rats at days 21 and 28, indicating the appearance of thermal hyperalgesia. Changes in expression of mRNAs for IL-1ß, TNF-α, MCP-1, MMP-9 and TIMP-1 were assessed using real-time polymerase chain reaction in spinal cord including the injury epicenter along with regions above and below the level of lesion at day 28 post-injury. A significant increase was observed in the expression of MCP-1, TNF-α, TIMP-1 and IL-1ß in the injury epicenter, whereas only TIMP-1 was upregulated in the area below the injury epicenter. The results of the study suggest that prolonged upregulation of inflammatory mediators might be involved in chronic neuropathic pain in SCI, and that TIMP-1 may play a role in maintenance of chronic below level pain.

Age-dependent response of CCAAT/enhancer binding proteins following traumatic brain injury in mice.

Exacerbated inflammatory responses have been reported following traumatic injury to the aged brain. The present study was designed to investigate the involvement of the transcription factors belonging to the CCAAT/enhancer binding protein (C/EBP) family that regulate expression of many of the pro-inflammatory genes which show increased expression following injury to the aged brain. Controlled cortical impact injury was induced in adult (5-6 months) and aged (22-24 months) C57/BL6 mice. C/EBP mRNA and protein expression were analyzed in injured cortex at 1, 3, and 7 days post-injury. Expression of C/EBPalpha was reduced relative to baseline at day 1 in both adult and aged mice, whereas, it increased at days 3 and 7 post-injury. No significant differences were observed between adult and aged brain. Upregulation of C/EBPbeta was observed 1 day following injury in both the adult and aged brain, but there were no major age-related differences in mRNA levels. However, there was higher C/EBPbeta protein in the aged brain. C/EBPdelta expression increased beginning 1 day post-injury in both adult and aged brain. In this case, the increase in C/EBPdelta expression was higher in the aged brain than in the adult at all time points studied. Expression of CCAAT/enhancer binding protein homologous protein (CHOP), a transcription factor involved in ER stress and protein unfolding responses, was also up-regulated in response to injury, but CHOP levels were significantly lower in the aged than the adult brain. Based on these results, we conclude that differential expression of C/EBP beta, delta and CHOP might contribute to the hyper-inflammatory response and poor prognosis following traumatic brain injury in the elderly patients. In addition elevated C/EBPdelta levels following TBI in the aged brain may play a role in the link between TBI and Alzheimer's disease.

Post-acute pathological changes in the thalamus and internal capsule in aged mice following controlled cortical impact injury: a magnetic resonance imaging, iron histochemical, and glial immunohistochemical study.

Traumatic brain injury (TBI) is a major cause of neurological disability across all ages, but the elderly are particularly vulnerable and have a worse prognosis than younger individuals. To advance the understanding of long-term pathogenesis induced by TBI in the elderly, aged mice (21 -- 24 months) were given a controlled cortical impact (CCI) injury to the sensorimotor cortex, and their brains were analyzed by MRI and histopathology at 1 and 2 months after CCI injury, a post-acute period. A T2 hypointensity was observed in the ipsilateral thalamus but not in the contralateral thalamus or in the thalamus of sham operated, control mice. The hypointensity was co-localized with increased histochemical staining of iron, a paramagnetic substance that causes a shortening of the T2 relaxation time. Since iron catalyzes reactions that lead to toxic free radicals, the deposition of iron in the thalamus raises the possibility that it promotes pathogenesis following TBI. Astrocyte gliosis and microgliosis were also observed in the ipsilateral thalamus in the post-acute period. The ipsilateral internal capsule displayed a trend for a T2 hypointensity, however, unlike the thalamus it did not have an increase of iron or GFAP staining, but it did have evidence of microgliosis. In summary, areas of T2 hypointensity were revealed in both the thalamus and internal capsule during the post-acute period following CCI injury, but the underlying pathology appeared to be distinct between these regions.

Impaired expression of neuroprotective molecules in the HIF-1alpha pathway following traumatic brain injury in aged mice.

Elderly traumatic brain injury (TBI) patients have higher rates of mortality and worse functional outcome than non-elderly TBI patients. The mechanisms involved in poor outcomes in the elderly are not well understood. Hypoxia-inducible factor-1 alpha (HIF-1alpha) is a basic helix-loop-helix transcription factor that modulates expression of key genes involved in neuroprotection. In this study, we studied the expression of HIF-1alpha and its target survival genes, heme oxygenase-1 (HO-1), vascular endothelial growth factor (VEGF), and erythropoietin (EPO) in the brains of adult versus aged mice following controlled cortical impact (CCI) injury. Adult (5-6 months) and aged (23-24 months) C57Bl/6 mice were injured using a CCI device. At 72 h post-injury, mice were sacrificed and the injured cortex was used for mRNA and protein analysis using real-time reverse transcription--polymerase chain reaction (RT-PCR) and Western blotting protocols. Following injury, HIF-1alpha, HO-1, and VEGF showed upregulation in both the young and aged mice, but in the aged animals the increase in HIF-1alpha and VEGF in response to injury was much lower than in the adult injured animals. EPO was upregulated in the adult injured brain, but not in the aged injured brain. These results support the hypothesis that reduced expression of genes in the HIF-1alpha neuroprotective pathway in aging may contribute to poor prognosis in the elderly following TBI.

Exacerbated glial response in the aged mouse hippocampus following controlled cortical impact injury.

Old age is associated with enhanced susceptibility to and poor recovery from brain injury. An exacerbated microglial and astrocyte response to brain injury might be involved in poor outcomes observed in the elderly. The present study was therefore designed to quantitate the expression of markers of microglia and astrocyte activation using real-time RT-PCR, immunoblot and immunohistochemical analysis in aging brain in response to brain injury. We examined the hippocampus, a region that undergoes secondary neuron death, in aged (21-24 months) and adult (5-6 months) mice following controlled cortical impact (CCI) injury to the sensorimotor cortex. Basal mRNA expression of CD11b and Iba1, markers of activated microglia, was higher in aged hippocampus as compared to the adult. The mRNA expression of microglial markers increased and reached maximum 3 days post-injury in both adult and aged mice, but was higher in the aged mice at all time points studied, and in the aged mice the return to baseline levels was delayed. Basal mRNA expression of GFAP and S100B, markers of activated astrocytes, was higher in aged mice. Both markers increased and reached maximum 7 days post-injury. The mRNA expression of astrocyte markers returned to near basal levels rapidly after injury in the adult mice, whereas again in the aged mice return to baseline was delayed. Immunochemical analysis using Iba1 and GFAP antibodies indicated accentuated glial responses in the aged hippocampus after injury. The pronounced and prolonged activation of microglia and astrocytes in hippocampus may contribute to worse cognitive outcomes in the elderly following TBI.

Detrimental effects of aging on outcome from traumatic brain injury: a behavioral, magnetic resonance imaging, and histological study in mice.

Considerable evidence indicates that outcomes from traumatic brain injury (TBI) are worse in the elderly, but there has been little preclinical research to explore potential mechanisms. In this study, we examined the age-related effects on outcome in a mouse model of controlled cortical impact (CCI) injury. We compared the responses of adult (5-6 months old) and aged (21-24 months old) male mice following a moderate lateral CCI injury to the sensorimotor cortex. Sensorimotor function was evaluated with the rotarod, gridwalk and spontaneous forelimb behavioral tests. Acute edema was assessed from hyperintensity on T2-weighted magnetic resonance images. Blood-brain barrier opening was measured using anti-mouse immunoglobulin G (IgG) immunohistochemistry. Neurodegeneration was assessed by amino-cupric silver staining, and lesion cavity volumes were measured from histological images. Indicators of injury were generally worse in the aged than the adult mice. Acute edema, measured at 24 and 48 h post-injury, resolved more slowly in the aged mice (p < 0.01). Rotarod recovery (p < 0.05) and gridwalk deficits (p < 0.01) were significantly worse in aged mice. There was greater (p < 0.01 at 3 days) and more prolonged post-acute opening of the blood-brain barrier in the aged mice. Neurodegeneration was greater in the aged mice (p < 0.01 at 3 days). In contrast, lesion cavity volumes, measured at 3 days post-injury, were not different between injured groups. These results suggest that following moderate controlled cortical impact injury, the aged brain is more vulnerable than the adult brain to neurodegeneration, resulting in greater loss of function. Tissue loss at the impact site does not explain the increased functional deficits seen in the aged animals. Prolonged acute edema, increased opening of the blood-brain barrier and increased neurodegeneration found in the aged animals implicate secondary processes in age-related differences in outcome.