Diacylglycerol Lipase-ß Knockout Mice Display a Sex-Dependent Attenuation of Traumatic Brain Injury-Induced Mortality with No Impact on Memory or Other Functional Consequences.

Background: The endogenous cannabinoid system modulates inflammatory signaling in a variety of pathological states, including traumatic brain injury (TBI). The selective expression of diacylglycerol lipase-ß (DAGL-ß), the 2-arachidonylglycerol biosynthetic enzyme, on resident immune cells of the brain (microglia) and the role of this pathway in neuroinflammation, suggest that this enzyme may contribute to TBI-induced neuroinflammation. Accordingly, we tested whether DAGL-ß-/- mice would show a protective phenotype from the deleterious consequences of TBI on cognitive and neurological motor functions. Materials and Methods: DAGL-ß-/- and -ß+/+ mice were subjected to the lateral fluid percussion model of TBI and assessed for learning and memory in the Morris water maze (MWM) Fixed Platform (reference memory) and Reversal (cognitive flexibility) tasks, as well as in a cued MWM task to infer potential sensorimotor/motivational deficits. In addition, subjects were assessed for motor behavior (Rotarod and the Neurological Severity Score assays) and in the light/dark box and the elevated plus maze to infer whether these manipulations affected anxiety-like behavior. Finally, we also examined whether brain injury disrupts the ceramide/sphingolipid lipid signaling system and if DAGL-ß deletion offers protection. Results: TBI disrupted all measures of neurological motor function and reduced body weight, but did not affect body temperature or performance in common assays used to infer anxiety. TBI also impaired performance in MWM Fixed Platform and Reversal tasks, but did not affect cued MWM performance. Although no differences were found between DAGL-ß-/- and -ß+/+ mice in any of these measures, male DAGL-ß-/- mice displayed an unexpected survival-protective phenotype, which persisted at increased injury severities. In contrast, TBI did not elicit mortality in female mice regardless of genotype. TBI also produced significant changes in sphingolipid profiles (a family of lipids, members of which have been linked to both apoptotic and antiapoptotic pathways), in which DAGL-ß deletion modestly altered levels of select species. Conclusions: These findings indicate that although DAGL-ß does not play a necessary role in TBI-induced cognitive and neurological function, it appears to contribute to the increased vulnerability of male mice to TBI-induced mortality, whereas female mice show high survival rates irrespective of DAGL-ß expression.

Time-dependent hemeoxygenase-1, lipocalin-2 and ferritin induction after non-contusion traumatic brain injury.

Traumatic brain injury (TBI) often presents with focal contusion and parenchymal bleeds, activating heme oxygenase (HO) to degrade released hemoglobin. Here we show that diffuse, midline fluid percussion injury causes time-dependent induction of HO-1 and iron binding proteins within both hemorrhagic neocortex and non-hemorrhagic hippocampus. Rats subjected to midline fluid percussion injury (FPI) survived 1-15d postinjury and tissue was collected for Western blot and immunohistochemical assays. HO-1 was elevated 1d after FPI, peaked at 3d, and returned to control baseline 7-15d. Iron management proteins lipocalin 2 (LCN2) and ferritin (FTL) exhibited distinct postinjury time courses, where peak LCN2 response preceded, and FTL followed that of HO-1. LCN2 elevation supported not only its role in iron transport, but also mediation of matrix metalloproteinase 9 (MMP9) activity. Upregulation of FTL for intracellular iron sequestration was delayed relative to both HO-1 and LCN2 induction. In the neocortex IBA-1+ microglia around the injury core expressed HO-1, but astrocytes co-localized with HO-1 in perilesional parenchyma. Non-hemorrhagic dentate gyrus showed predominant HO-1 labeling in hilar microglia and in molecular layer astrocytes. At 1d postinjury, LCN2 and HO-1 co-localized in a subpopulation of reactive glia within both brain regions. Notably, FTL was distributed within cells around injured vessels, damaged subcortical white matter, and along vessels of the hippocampal fissure. Together these results confirm that even the moderate, non-contusional insult of diffuse midline FPI can significantly activate postinjury HO-1 heme processing pathways and iron management proteins. Moreover, this activation is time-dependent and occurs in the absence of overt hemorrhage.

Matrix metalloproteinase signals following neurotrauma are right on cue.

Neurotrauma, a term referencing both traumatic brain and spinal cord injuries, is unique to neurodegeneration in that onset is clearly defined. From the perspective of matrix metalloproteinases (MMPs), there is opportunity to define their temporal participation in injury and recovery beginning at the level of the synapse. Here we examine the diverse roles of MMPs in the context of targeted insults (optic nerve lesion and hippocampal and olfactory bulb deafferentation), and clinically relevant focal models of traumatic brain and spinal cord injuries. Time-specific MMP postinjury signaling is critical to synaptic recovery after focal axonal injuries; members of the MMP family exhibit a signature temporal profile corresponding to axonal degeneration and regrowth, where they direct postinjury reorganization and synaptic stabilization. In both traumatic brain and spinal cord injuries, MMPs mediate early secondary pathogenesis including disruption of the blood-brain barrier, creating an environment that may be hostile to recovery. They are also critical players in wound healing including angiogenesis and the formation of an inhibitory glial scar. Experimental strategies to reduce their activity in the acute phase result in long-term neurological recovery after neurotrauma and have led to the first clinical trial in spinal cord injured pet dogs.

Matrix Metalloproteinase 9 and Osteopontin Interact to Support Synaptogenesis in the Olfactory Bulb after Mild Traumatic Brain Injury.

Olfactory receptor axons reinnervate the olfactory bulb (OB) after chemical or transection lesion. Diffuse brain injury damages the same axons, but the time course and regulators of OB reinnervation are unknown. Gelatinases (matrix metalloproteinase [MMP]2, MMP9) and their substrate osteopontin (OPN) are candidate mediators of synaptogenesis after central nervous system (CNS) insult, including olfactory axon damage. Here, we examined the time course of MMP9, OPN, and OPN receptor CD44 response to diffuse OB injury. FVBV/NJ mice received mild midline fluid percussion insult (mFPI), after which MMP9 activity and both OPN and CD44 protein expression were measured. Diffuse mFPI induced time-dependent increase in OB MMP9 activity and elevated the cell signaling 48-kD OPN fragment. This response was bimodal at 1 and 7 days post-injury. MMP9 activity was also correlated with 7-day reduction in a second 32-kD OPN peptide. CD44 increase peaked at 3 days, delayed relative to MMP9/OPN response. MMP9 and OPN immunohistochemistry suggested that deafferented tufted and mitral neurons were the principal sites for these molecular interactions. Analysis of injured MMP9 knockout (KO) mice showed that 48-kD OPN production was dependent on OB MMP9 activity, but with no KO effect on CD44 induction. Olfactory marker protein (OMP), used to identify injured olfactory axons, revealed persistent axon damage in the absence of MMP9. MMP9 KO ultrastructure at 21 days post-injury indicated that persistent OMP reduction was paired with delayed removal of degenerated axons. These results provide evidence that diffuse, concussive brain trauma induces a post-injury interaction between MMP9, OPN, and CD44, which mediates synaptic plasticity and reinnervation within the OB.

Sex Differences in Synaptic Plasticity: Hormones and Beyond.

Notable sex-differences exist between neural structures that regulate sexually dimorphic behaviors such as reproduction and parenting. While anatomical differences have been well-characterized, advancements in neuroimaging and pharmacology techniques have allowed researchers to identify differences between males and females down to the level of the synapse. Disparate mechanisms at the synaptic level contribute to sex-specific neuroplasticity that is reflected in sex-dependent behaviors. Many of these synaptic differences are driven by the endocrine system and its impact on molecular signaling and physiology. While sex-dependent modifications exist at baseline, further differences emerge in response to stimuli such as stressors. While some of these mechanisms are unifying between sexes, they often have directly opposing consequences in males and females. This variability is tied to gonadal steroids and their interactions with intra- and extra-cellular signaling mechanisms. This review article focuses on the various mechanisms by which sex can alter synaptic plasticity, both directly and indirectly, through steroid hormones such as estrogen and testosterone. That sex can drive neuroplasticity throughout the brain, highlights the importance of understanding sex-dependent neural mechanisms of the changing brain to enhance interpretation of results regarding males and females. As mood and stress responsivity are characterized by significant sex-differences, understanding the molecular mechanisms that may be altering structure and function can improve our understanding of these behavioral and mental characteristics.

Mild Fluid Percussion Injury Induces Diffuse Axonal Damage and Reactive Synaptic Plasticity in the Mouse Olfactory Bulb.

Despite the regenerative capacity of the olfactory bulb (OB), head trauma causes olfactory disturbances in up to 30% of patients. While models of olfactory nerve transection, olfactory receptor neuron (ORN) ablation, or direct OB impact have been used to examine OB recovery, these models are severe and not ideal for study of OB synaptic repair. We posited that a mild fluid percussion brain injury (mFPI), delivered over mid-dorsal cortex, would produce diffuse OB deafferentation without confounding pathology. Wild type FVB/NJ mice were subjected to mFPI and OB probed for ORN axon degeneration and onset of reactive synaptogenesis. OB extracts revealed 3 d postinjury elevation of calpain-cleaved 150-kDa αII-spectrin, an indicator of axon damage, in tandem with reduced olfactory marker protein (OMP), a protein specific to intact ORN axons. Moreover, mFPI also produced a 3-d peak in GFAP+ astrocyte and IBA1+ microglial reactivity, consistent with postinjury inflammation. OB glomeruli showed disorganized ORN axons, presynaptic degeneration, and glial phagocytosis at 3 and 7 d postinjury, all indicative of deafferentation. At 21 d after mFPI, normal synaptic structure re-emerged along with OMP recovery, supporting ORN afferent reinnervation. Robust 21 d postinjury upregulation of GAP-43 was consistent with the time course of ORN axon sprouting and synapse regeneration reported after more severe olfactory insult. Together, these findings define a cycle of synaptic degeneration and recovery at a site remote to non-contusive brain injury. We show that mFPI models diffuse ORN axon damage, useful for the study of time-dependent reactive synaptogenesis in the deafferented OB.

Investigation of left and right lateral fluid percussion injury in C57BL6/J mice: In vivo functional consequences.

Although rodent models of traumatic brain injury (TBI) reliably produce cognitive and motor disturbances, behavioral characterization resulting from left and right hemisphere injuries remains unexplored. Here we examined the functional consequences of targeting the left versus right parietal cortex in lateral fluid percussion injury, on Morris water maze (MWM) spatial memory tasks (fixed platform and reversal) and neurological motor deficits (neurological severity score and rotarod). In the MWM fixed platform task, right lateral injury produced a small delay in acquisition rate compared to left. However, injury to either hemisphere resulted in probe trial deficits. In the MWM reversal task, left-right performance deficits were not evident, though left lateral injury produced mild acquisition and probe trial deficits compared to sham controls. Additionally, left and right injury produced similar neurological motor task deficits, impaired righting times, and lesion volumes. Injury to either hemisphere also produced robust ipsilateral, and modest contralateral, morphological changes in reactive microglia and astrocytes. In conclusion, left and right lateral TBI impaired MWM performance, with mild fixed platform acquisition rate differences, despite similar motor deficits, histological damage, and glial cell reactivity. Thus, while both left and right lateral TBI produce cognitive deficits, laterality in mouse MWM learning and memory merits consideration in the investigation of TBI-induced cognitive consequences.

Targeting Kv1.3 channels to reduce white matter pathology after traumatic brain injury.

Axonal injury is present in essentially all clinically significant cases of traumatic brain injury (TBI). While no effective treatment has been identified to date, experimental TBI models have shown promising axonal protection using immunosuppressants FK506 and Cyclosporine-A, with treatment benefits attributed to calcineurin inhibition or protection of mitochondrial function. However, growing evidence suggests neuroprotective efficacy of these compounds may also involve direct modulation of ion channels, and in particular Kv1.3. The present study tested whether blockade of Kv1.3 channels, using Clofazimine (CFZ), would alleviate TBI-induced white matter pathology in rodents. Postinjury CFZ administration prevented suppression of compound action potential (CAP) amplitude in the corpus callosum of adult rats following midline fluid percussion TBI, with injury and treatment effects primarily expressed in unmyelinated CAPs. Kv1.3 protein levels in callosal tissue extracts were significantly reduced postinjury, but this loss was prevented by CFZ treatment. In parallel, CFZ also attenuated the injury-induced elevation in pro-inflammatory cytokine IL1-ß. The effects of CFZ on glial function were further studied using mixed microglia/astrocyte cell cultures derived from P3-5 mouse corpus callosum. Cultures of callosal glia challenged with lipopolysaccharide exhibited a dramatic increase in IL1-ß levels, accompanied by reactive morphological changes in microglia, both of which were attenuated by CFZ treatment. These results support a cell specific role for Kv1.3 signaling in white matter pathology after TBI, and suggest a treatment approach based on the blockade of these channels. This therapeutic strategy may be especially efficacious for normalizing neuro-glial interactions affecting unmyelinated axons after TBI.

Time dependent integration of matrix metalloproteinases and their targeted substrates directs axonal sprouting and synaptogenesis following central nervous system injury.

Over the past two decades, many investigators have reported how extracellular matrix molecules act to regulate neuroplasticity. The majority of these studies involve proteins which are targets of matrix metalloproteinases. Importantly, these enzyme/substrate interactions can regulate degenerative and regenerative phases of synaptic plasticity, directing axonal and dendritic reorganization after brain insult. The present review first summarizes literature support for the prominent role of matrix metalloproteinases during neuroregeneration, followed by a discussion of data contrasting adaptive and maladaptive neuroplasticity that reveals time-dependent metalloproteinase/substrate regulation of postinjury synaptic recovery. The potential for these enzymes to serve as therapeutic targets for enhanced neuroplasticity after brain injury is illustrated with experiments demonstrating that metalloproteinase inhibitors can alter adaptive and maladaptive outcome. Finally, the complexity of metalloproteinase role in reactive synaptogenesis is revealed in new studies showing how these enzymes interact with immune molecules to mediate cellular response in the local regenerative environment, and are regulated by novel binding partners in the brain extracellular matrix. Together, these different examples show the complexity with which metalloproteinases are integrated into the process of neuroregeneration, and point to a promising new angle for future studies exploring how to facilitate brain plasticity.

Osteopontin expression in acute immune response mediates hippocampal synaptogenesis and adaptive outcome following cortical brain injury.

Traumatic brain injury (TBI) produces axotomy, deafferentation and reactive synaptogenesis. Inflammation influences synaptic repair, and the novel brain cytokine osteopontin (OPN) has potential to support axon regeneration through exposure of its integrin receptor binding sites. This study explored whether OPN secretion and proteolysis by matrix metalloproteinases (MMPs) mediate the initial degenerative phase of synaptogenesis, targeting reactive neuroglia to affect successful repair. Adult rats received unilateral entorhinal cortex lesion (UEC) modeling adaptive synaptic plasticity. Over the first week postinjury, hippocampal OPN protein and mRNA were assayed and histology was performed. At 1-2d, OPN protein increased up to 51 fold, and was localized within activated, mobilized glia. OPN transcript also increased over 50 fold, predominantly within reactive microglia. OPN fragments known to be derived from MMP proteolysis were elevated at 1d, consistent with prior reports of UEC glial activation and enzyme production. Postinjury minocycline immunosuppression attenuated MMP-9 gelatinase activity, which was correlated with the reduction of neutrophil gelatinase-associated lipocalin (LCN2) expression, and reduced OPN fragment generation. The antibiotic also attenuated removal of synapsin-1 positive axons from the deafferented zone. OPN KO mice subjected to UEC had similar reduction of hippocampal MMP-9 activity, as well as lower synapsin-1 breakdown over the deafferented zone. MAP1B and N-cadherin, surrogates of cytoarchitecture and synaptic adhesion, were not affected. OPN KO mice with UEC exhibited time dependent cognitive deficits during the synaptogenic phase of recovery. This study demonstrates that OPN can mediate immune response during TBI synaptic repair, positively influencing synapse reorganization and functional recovery.

MT5-MMP, ADAM-10, and N-cadherin act in concert to facilitate synapse reorganization after traumatic brain injury.

Matrix metalloproteinases (MMPs) influence synaptic recovery following traumatic brain injury (TBI). Membrane type 5-matrix metalloproteinase (MT5-MMP) and a distintegrin and metalloproteinase-10 (ADAM-10) are membrane-bound MMPs that cleave N-cadherin, a protein critical to synapse stabilization. This study examined protein and mRNA expression of MT5-MMP, ADAM-10, and N-cadherin after TBI, contrasting adaptive and maladaptive synaptogenesis. The effect of MMP inhibition on MT5-MMP, ADAM-10, and N-cadherin was assessed during maladaptive plasticity and correlated with synaptic function. Rats were subjected to adaptive unilateral entorhinal cortical lesion (UEC) or maladaptive fluid percussion TBI+bilateral entorhinal cortical lesion (TBI+BEC). Hippocampal MT5-MMP and ADAM-10 protein was significantly elevated 2 and 7 days post-injury. At 15 days after UEC, each MMP returned to control level, while TBI+BEC ADAM-10 remained elevated. At 2 and 7 days, N-cadherin protein was below control. By the 15-day synapse stabilization phase, UEC N-cadherin rose above control, a shift not seen for TBI+BEC. At 7 days, increased TBI+BEC ADAM-10 transcript correlated with protein elevation. UEC ADAM-10 mRNA did not change, and no differences in MT5-MMP or N-cadherin mRNA were detected. Confocal imaging showed MT5-MMP, ADAM-10, and N-cadherin localization within reactive astrocytes. MMP inhibition attenuated ADAM-10 protein 15 days after TBI+BEC and increased N-cadherin. This inhibition partially restored long-term potentiation induction, but did not affect paired-pulse facilitation. Our results confirm time- and injury-dependent expression of MT5-MMP, ADAM-10, and N-cadherin during reactive synaptogenesis. Persistent ADAM-10 expression was correlated with attenuated N-cadherin level and reduced functional recovery. MMP inhibition shifted ADAM-10 and N-cadherin toward adaptive expression and improved synaptic function.

Unmyelinated axons show selective rostrocaudal pathology in the corpus callosum after traumatic brain injury.

Axonal injury is consistently observed after traumatic brain injury (TBI). Prior research has extensively characterized the post-TBI response in myelinated axons. Despite evidence that unmyelinated axons comprise a numerical majority of cerebral axons, pathologic changes in unmyelinated axons after TBI have not been systematically studied. To identify morphologic correlates of functional impairment of unmyelinated fibers after TBI, we assessed ultrastructural changes in corpus callosum axons. Adult rats received moderate fluid percussion TBI, which produced diffuse injury with no contusion. Cross-sectional areas of 13,797 unmyelinated and 3,278 intact myelinated axons were stereologically measured at survival intervals from 3 hours to 15 days after injury. The mean caliber of unmyelinated axons was significantly reduced at 3 to 7 days and recovered by 15 days, but the time course of this shrinkage varied among the genu, mid callosum, and splenium. Relatively large unmyelinated axons seemed to be particularly vulnerable. Injury-induced decreases in unmyelinated fiber density were also observed, but they were more variable than caliber reductions. By contrast, no significant morphometric changes were observed in myelinated axons. The finding of a preferential vulnerability in unmyelinated axons has implications for current concepts of axonal responses after TBI and for development of specifically targeted therapies.

Phosphacan and receptor protein tyrosine phosphatase ß expression mediates deafferentation-induced synaptogenesis.

This study documents the spatial and temporal expression of three structurally related chondroitin sulfated proteoglycans (CSPGs) during synaptic regeneration induced by brain injury. Using the unilateral entorhinal cortex (EC) lesion model of adaptive synaptogenesis, we documented mRNA and protein profiles of phosphacan and its two splice variants, full length receptor protein tyrosine phosphatase ß (RPTPß) and the short transmembrane receptor form (sRPTPß), at 2, 7, and 15 days postlesion. We report that whole hippocampal sRPTPß protein and mRNA are persistently elevated over the first two weeks after UEC. As predicted, this transmembrane family member was localized adjacent to synaptic sites in the deafferented neuropil and showed increased distribution over that zone following lesion. By contrast, whole hippocampal phosphacan protein was not elevated with deafferentation; however, its mRNA was increased during the period of sprouting and synapse formation (7d). When the zone of synaptic reorganization was sampled using molecular layer/granule cell (ML/GCL) enriched dissections, we observed an increase in phosphacan protein at 7d, concurrent with the observed hippocampal mRNA elevation. Immunohistochemistry also showed a shift in phosphacan distribution from granule cell bodies to the deafferented ML at 2 and 7d postlesion. Phosphacan and sRPTPß were not colocalized with glial fibrillary acid protein (GFAP), suggesting that reactive astrocytes were not a major source of either proteoglycan. While transcript for the developmentally prominent full length RPTPß was also increased at 2 and 15d, its protein was not detected in our adult samples. These results indicate that phosphacan and RPTPß splice variants participate in both the acute degenerative and long-term regenerative phases of reactive synaptogenesis. These results suggest that increase in the transmembrane sRPTPß tyrosine phosphatase activity is critical to this plasticity, and that local elevation of extracellular phosphacan influences dendritic organization during synaptogenesis.

Proteolysis of submembrane cytoskeletal proteins ankyrin-G and αII-spectrin following diffuse brain injury: a role in white matter vulnerability at Nodes of Ranvier.

A high membrane-to-cytoplasm ratio makes axons particularly vulnerable to traumatic injury. Posttraumatic shifts in ionic homeostasis promote spectrin cleavage, disrupt ankyrin linkages and destabilize axolemmal proteins. This study contrasted ankyrin-G and αII-spectrin degradation in cortex and corpus callosum following diffuse axonal injury produced by fluid percussion insult. Ankyrin-G lysis occurred preferentially in white matter, with acute elevation of all fragments and long-term reduction of a low kD form. Calpain-generated αII-spectrin fragments increased in both regions. Caspase-3 lysis of αII-spectrin showed a small, acute rise in cortex but was absent in callosum. White matter displayed nodal damage, with horseradish peroxidase permeability into the submyelin space. Ankyrin-G-binding protein neurofascin and spectrin-binding protein ankyrin-B showed acute alterations in expression. These results support ankyrin-G vulnerability in white matter following trauma and suggest that ankyrin-G and αII-spectrin proteolysis disrupts Node of Ranvier integrity. The time course of such changes were comparable to previously observed functional deficits in callosal fibers.

The effects of cyclosporin-A on axonal conduction deficits following traumatic brain injury in adult rats.

Immunophilin ligands, including cyclosporin-A (CsA), have been shown to be neuroprotective in experimental models of traumatic brain injury (TBI) and to attenuate the severity of traumatic axonal injury. Prior studies have documented CsA treatment to reduce essential components of posttraumatic axonal pathology, including impaired axoplasmic transport, spectrin proteolysis, and axonal swelling. However, the effects of CsA administration on axonal function, following TBI, have not been evaluated. The present study assessed the effects of CsA treatment on compound action potentials (CAPs) evoked in corpus callosum of adult rats following midline fluid percussion injury. Rats received a 20 mg/kg bolus of CsA, or cremaphor vehicle, at either 15 min or 1 h postinjury, and at 24 h postinjury CAP recording was conducted in coronal brain slices. To elucidate how injury and CsA treatments affect specific populations of axons, CAP waveforms generated largely by myelinated axons (N1) were analyzed separately from the CAP signal, which predominantly reflects activity in unmyelinated axons (N2). CsA administration at 15 min postinjury resulted in significant protection of CAP area, and this effect was more pronounced in N1, than in the N2, CAP component. This treatment also significantly protected against TBI-induced reductions in high-frequency responding of the N1 CAP signal. In contrast, CsA treatment at 1 h did not significantly protect CAPs but was associated with atypical waveforms in N1 CAPs, including decreased CAP duration and reduced refractoriness. The present findings also support growing evidence that myelinated and unmyelinated axons respond differentially to injury and neuroprotective compounds.

Injury modality, survival interval, and sample region are critical determinants of qRT-PCR reference gene selection during long-term recovery from brain trauma.

In the present study we examined expression of four real-time quantitative RT-PCR reference genes commonly applied to rodent models of brain injury. Transcripts for beta-actin, cyclophilin A, GAPDH, and 18S rRNA were assessed at 2-15 days post-injury, focusing on the period of synaptic recovery. Diffuse moderate central fluid percussion injury (FPI) was contrasted with unilateral entorhinal cortex lesion (UEC), a model of targeted deafferentation. Expression in UEC hippocampus, as well as in FPI hippocampus and parietotemporal cortex was analyzed by qRT-PCR. Within-group variability of gene expression was assessed and change in expression relative to paired controls was determined. None of the four common reference genes tested was invariant across brain region, survival time, and type of injury. Cyclophilin A appeared appropriate as a reference gene in UEC hippocampus, while beta-actin was most stable for the hippocampus subjected to FPI. However, each gene may fail as a suitable reference with certain test genes whose RNA expression is targeted for measurement. In FPI cortex, all reference genes were significantly altered over time, compromising their utility for time-course studies. Despite such temporal variability, certain genes may be appropriate references if limited to single survival times. These data provide an extended baseline for identification of appropriate reference genes in rodent studies of recovery from brain injury. In this context, we outline additional considerations for selecting a qRT-PCR normalization strategy in such studies. As previously concluded for acute post-injury intervals, we stress the importance of reference gene validation for each brain injury paradigm and each set of experimental conditions.

Increased MMP-3 and CTGF expression during lipopolysaccharide-induced dopaminergic neurodegeneration.

Accumulating evidence indicates that neuroinflammation contributes significantly to progressive dopaminergic (DA) neurodegeneration in Parkinson's disease (PD). Altered matrix metalloproteinase-3 (MMP-3) expression has been reported in several neuroinflammatory paradigms; however, its relationship to inflammation-induced DA neurotoxicity has not been explored. To this end, we investigated the temporal expression pattern of MMP-3 and one of its downstream targets, connective tissue growth factor (CTGF), following lipopolysaccharide (LPS)-induced DA neurodegeneration. LPS was directly injected into the substantia nigra of male Sprague-Dawley rats. Lesion formation was confirmed with immunohistochemistry 48 h post-injection. MMP-3 and CTGF were measured by western blot 12, 24, and 48 h post-injection. In association with neurodegeneration, MMP-3 expression and activation was significantly increased 24 and 48 h after LPS injection. In addition, CTGF expression increased 5-fold at the 24h time point. The temporal changes in MMP-3 and CTGF expression corresponded to the neurodegenerative phase of this model, suggesting that these two proteins may participate in neuroinflammation-induced DA neurotoxicity.

Agrin expression during synaptogenesis induced by traumatic brain injury.

Interaction between extracellular matrix proteins and regulatory proteinases can mediate synaptic integrity. Previously, we documented that matrix metalloproteinase 3 (MMP-3) expression and activity increase following traumatic brain injury (TBI). We now report protein and mRNA analysis of agrin, a MMP-3 substrate, over the time course of trauma-induced synaptogenesis. Agrin expression during the successful synaptic reorganization of unilateral entorhinal cortical lesion (UEC) was compared with expression when normal synaptogenesis fails (combined fluid percussion TBI and bilateral entorhinal lesion [BEC]). We observed that agrin protein was increased in both models at 2 and 7 days postinjury, and immuohistochemical (IHC) co-localization suggested reactive astrocytes contribute to that increase. Agrin formed defined boundaries for sprouting axons along deafferented dendrites in the UEC, but failed to do so after combined insult. Similarly, Western blot analysis revealed greater increase in UEC agrin protein relative to the combined TBI+BEC model. Both models showed increased agrin transcription at 7 days postinjury and mRNA normalization by 15 days. Attenuation of synaptic pathology with the NMDA antagonist MK-801 reduced 7-day UEC agrin transcript to a level not different from unlesioned controls. By contrast, MK-801 in the combined insult failed to significantly change 7-day agrin transcript, mRNA levels remaining elevated over uninjured sham cases. Together, these results suggest that agrin plays an important role in the sprouting phase of reactive synaptogenesis, and that both its expression and distribution are correlated with extent of successful recovery after TBI. Further, when pathogenic conditions which induce synaptic plasticity are reduced, increase in agrin mRNA is attenuated.

Preferential neuroprotective effect of tacrolimus (FK506) on unmyelinated axons following traumatic brain injury.

Prior investigations of traumatic axonal injury (TAI), and pharmacological treatments of TAI pathology, have focused exclusively on the role of myelinated axons, with no systematic observations directed towards unmyelinated axon pathophysiology. Recent electrophysiological evidence, however, indicates that unmyelinated axons are more vulnerable than myelinated axons in a rodent model of experimental TAI. Given their susceptibility to TAI, the present study examines whether unmyelinated axons also respond differentially to FK506, an immunophilin ligand with well-established neuroprotective efficacy in the myelinated fiber population. Adult rats received 3.0 mg/kg FK506 intravenously at 30 min prior to midline fluid percussion injury. In brain slice electrophysiological recordings, conducted at 24 h postinjury, compound action potentials (CAPs) were evoked in the corpus callosum, and injury effects quantified separately for CAP waveform components generated by myelinated axons (N1 wave) and unmyelinated axons (N2 wave). The amplitudes of both CAP components were suppressed postinjury, although this deficit was 16% greater for the N2 CAP. While FK506 treatment provided significant neuroprotection for both N1 and N2 CAPs, the drug benefit for the N2 CAP amplitude was 122% greater than that for the N1 CAPs, and improved postinjury strength-duration and refractoriness properties only in N2 CAPs. Immunocytochemical observations, of TAI reflected in intra-axonal pooling of amyloid precursor protein, indicated that FK506 reduced the extent of postinjury impairments to axonal transport and subsequent axonal damage. Collectively, these studies further substantiate a distinctive role of unmyelinated axons in TAI, and suggest a highly efficacious neuroprotective strategy to target this axonal population.

Myelinated and unmyelinated axons of the corpus callosum differ in vulnerability and functional recovery following traumatic brain injury.

Traumatic axonal injury (TAI), a common feature of traumatic brain injury, is associated with postinjury morbidity and mortality. However, TAI is not uniformly expressed in all axonal populations, with fiber caliber and anatomical location influencing specific TAI pathology. To study differential axonal vulnerability to brain injury, axonal excitability and integrity were assessed in the corpus callosum following fluid percussion injury in the rat. In brain slice electrophysiological recordings, compound action potentials (CAPs) were evoked in the corpus callosum, and injury effects were quantified separately for CAP waveform components generated by myelinated axons (N1 wave) and by unmyelinated axons (N2 wave). Ultrastructural analyses were also conducted of TAI-induced morphological changes in these axonal populations. The two populations of axons differed in response to brain injury, and in their functional recovery, during the first week postinjury. Amplitudes of N1 and N2 were significantly depressed at 3 h, 1 day, and 3 days survival. N1 amplitudes exhibited a recovery to control levels by 7 days postinjury. In contrast, N2 amplitudes were persistently suppressed through 7 days postinjury. Strength-duration properties of evoked CAPs further differentiated the effects of injury in these axonal populations, with N2 exhibiting an elevated strength-duration time constant postinjury. Ultrastructural observations revealed degeneration of myelinated axons consistent with diffuse injury sequelae, as well as previously undocumented pathology within the unmyelinated fiber population. Collectively, these findings demonstrate differential vulnerabilities of axons to brain injury and suggest that damage to unmyelinated fibers may play a significant role in morbidity associated with brain injury.