The expression of the protooncogenes, c-fos, jun B, c-jun, and jun D was investigated in a rat focal cerebral ischemia model by Northern analysis and in situ hybridization. Severe ischemia (reduction of regional blood flow by 88-92%) in this model is confined to cerebral cortex irrigated by the right middle cerebral artery. Ischemia for 30 minutes, which caused only slight cortical damage (infarct size, < 10 mm3), induced both jun B and c-fos mRNAs exclusively in the right cerebral cortex. Ischemia for 90 minutes, which led to large cortical infarction (infarct size, > 140 mm3), also induced the expression of these two genes in the right cerebral cortex as well as the ipsilateral hippocampus. The latter sustained very mild ischemia (reduction of regional blood flow by 10-20%). The coinduction of jun B and c-fos expression occurred immediately after reperfusion and peaked at 60 minutes after reperfusion. The expression of c-jun was enhanced in a similar pattern, but at a much lower magnitude. In contrast, no change in jun D expression was observed. Nuclear run-on assays indicated that the increase in c-fos, jun B, and c-jun mRNA levels was due to the increase of transcription rate in these genes. Mobility shift assays showed a basal DNA binding activity of transcription factor AP-1 in the right cerebral cortex. Ischemia for 30 or 90 minutes followed by reperfusion for 4 hours resulted in a four- to sixfold increase of AP-1 binding activity. The enhanced DNA binding activity persisted for as long as 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

a. 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 CGRP and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors. b. 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 (PBS) to the dura were measured to model changes in routine activity and allodynia. Calcitonin gene related peptide (CGRP), an important mediator of migraine pathogenesis, and the three components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein (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. c. Methods Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 microliter 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 min 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. d. 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, ...

The present study establishes a new mouse model for traumatic brain injury (TBI), using an electromechanically driven linear motor impactor device to deliver a lateral controlled cortical impact (CCI) injury to the sensorimotor cortex. Lesion cavity size was measured, and inter-animal consistency demonstrated, at 14 days post injury. Qualitative information regarding damage progression over time was obtained by scanning with high field magnetic resonance imaging (MRI) at five time points following injury. Functional impairment and recovery were measured with the Rotarod, gridwalk and cylinder tests, and lesion cavity volume was measured post mortem with thionin-stained tissue sections. The study establishes the reliability of a linear-motor based device for producing repeatable damage in a CCI model, demonstrates the power of longitudinal MRI in studying damage evolution, and confirms that a simple battery of functional tests record sensorimotor impairment and recovery.

Activation of c-fos, an immediate early gene, and the subsequent expression of the Fos protein have been noted following focal cerebral ischemia. Fos and Jun form a heterodimer as activator protein 1 (AP-1), which transregulates the expression of several genes. To study the postischemic events related to c-fos expression, we suppressed the expression of c-fos by intraventricular infusion of an antisense Oligodeoxynucleotide (anti-rncfosr 115 ) of c-fos mRNA. The effectiveness of antirncfosr 115 was confirmed first by its capability to block in vitro c-fos mRNA translation. In vivo, after intraventricular infusion of 32 P-labeled anti-rncfosr 115 , the oligodeoxynucleotide was internalized within 6 hours and detectable also in the nucleic acids fraction up to 41 hours. Treatment of the recovered nucleic acids with RNase H separated the labeled Oligodeoxynucleotide from the nucleic acid fraction, indicating an association of the antisense Oligodeoxynucleotide and cellular RNA after uptake. When focal cerebral ischemia was induced 16 hours after the infusion of antirncfosr 115 , the postischemic increase in Fos expression and AP-1 binding activity were suppressed. Specificity of the effect of anti-rncfosr 115 was suggested by its failure to suppress the DNA binding activity of nuclear cyclic AMP response elements. These results support the hypothesis that increased AP-1 binding activity following focal cerebral ischemia is dependent on Fos expression and can be inhibited in vivo by antisense c-fos oligodeoxynucleotides.The molecular events of brain adaptation to injury that may underlie functional recovery after stroke remain largely undefined. Recent observations of altered gene expression in ischemic brain using animal stroke models have opened new avenues for exploration of the biochemical cascades after stroke [1][2][3][4][5][6][7][8][9][10][11]. These postischemic events include an increase in extracellular excitatory amino acid neurotransmitters such as glutamate. Glutamate receptor-mediated activation of phospholipases and protein kinases results in the alteration of nuclear regulatory processes, including the expression of immediate early genes such as c-fos, junB, and c-jun [5,12]. The Fos, Jun, and JunB proteins have been shown to form activator protein 1 (AP-1) through a conserved dimerization domain, i.e., the leucine zipper [13]. Transcription regulator AP-1 protein binds a specific DNA motif and is believed to transactivate the expression of a number of late effector genes [14][15][16][17][18][19]. In the ischemic brain, we have previously demonstrated an increase in AP-1 binding activity [9]. A number of genes that bear neurotrophic properties may be regulated by transcription regulator AP-1. These genes, such as heat shock protein [1,4,[19][20][21][22], amyloid [23], neurotrophins [7], and protein tyrosine kinase receptor trkB [24], have been shown to be induced following cerebral ischemia. The causal relationship between the Fos/Jun-AP-1 cascade and the subsequent expression of the late e...

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-1β, 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-1β, 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-1β 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.

We have examined free radical production in a rat model of focal cerebral ischemia using microdialysis coupled with EPR analysis. A microdialysis probe was inserted 2 mm into the cerebral cortex, supplied by the right middle cerebral artery (MCA), and after a 2-hour washout period with artificial cerebral spinal fluid (ACSF), the perfusate solution was changed to ACSF containing the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). No free radicals were detected by DMPO during the pre-ischemia period. Both common carotid arteries and the right MCA were then ligated for 90 minutes. Microdialysate collected every 15 min during the ischemic period demonstrated predominantly superoxide or peroxyl radical production. After release of the occlusive sutures, hydroxyl radical became apparent initially, then thiyl and carbon centered radicals appeared later in samples collected every 15 min for two hours following cortical reperfusion. Careful studies on the purification and stability of DMPO solution were performed to circumvent artifacts and spurious signals.

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