Repetitive mild traumatic brain injury with impact acceleration in the mouse: Multifocal axonopathy, neuroinflammation, and neurodegeneration in the visual system.

Repetitive mild traumatic brain injury (mTBI) is implicated in chronic neurological illness. The development of animal models of repetitive mTBI in mice is essential for exploring mechanisms of these chronic diseases, including genetic vulnerability by using transgenic backgrounds. In this study, the rat model of impact acceleration (IA) was redesigned for the mouse cranium and used in two clinically relevant repetitive mTBI paradigms. We first determined, by using increments of weight dropped from 1m that the 40g weight was most representative of mTBI and was not associated with fractures, brain contusions, anoxic-ischemic injury, mortality, or significant neurological impairments. Quantitative evaluation of traumatic axonal injury (TAI) in the optic nerve/tract, cerebellum and corpus callosum confirmed that weight increase produced a graded injury. We next evaluated two novel repetitive mTBI paradigms (1 time per day or 3 times per day at days 0, 1, 3, and 7) and compared the resulting TAI, neuronal cell death, and neuroinflammation to single hit mTBI at sub-acute (7days) and chronic time points (10weeks) post-injury. Both single and repetitive mTBI caused TAI in the optic nerve/tract, cerebellum, corticospinal tract, lateral lemniscus and corpus callosum. Reactive microglia with phagocytic phenotypes were present at injury sites. Severity of axonal injury corresponded to impact load and frequency in the optic nerve/tract and cerebellum. Both single and repeat injury protocols were associated with retinal ganglion cell loss and optic nerve degeneration; these outcomes correlated with impact load and number/frequency. No phosphorylated tau immunoreactivity was detected in the brains of animals subjected to repetitive mTBI. Our findings establish a new model of repetitive mTBI model featured by TAI in discrete CNS tracts, especially the visual system and cerebellum. Injury in retina and optic nerve provides a sensitive measure of severity of mTBI, thus enabling further studies on mechanisms and experimental therapeutics. Our model can also be useful in exploring mechanisms of chronic neurological disease caused by repetitive mTBI in wild-type and transgenic mice.

Selective vasopressin-1a receptor antagonist prevents brain edema, reduces astrocytic cell swelling and GFAP, V1aR and AQP4 expression after focal traumatic brain injury.

A secondary and often lethal consequence of traumatic brain injury is cellular edema that we posit is due to astrocytic swelling caused by transmembrane water fluxes augmented by vasopressin-regulated aquaporin-4 (AQP4). We therefore tested whether vasopressin 1a receptor (V1aR) inhibition would suppress astrocyte AQP4, reduce astrocytic edema, and thereby diminish TBI-induced edematous changes. V1aR inhibition by SR49059 significantly reduced brain edema after cortical contusion injury (CCI) in rat 5h post-injury. Injured-hemisphere brain water content (n=6 animals/group) and astrocytic area (n=3/group) were significantly higher in CCI-vehicle (80.5±0.3%; 18.0±1.4 µm(2)) versus sham groups (78.3±0.1%; 9.5±0.9 µm(2)), and SR49059 blunted CCI-induced increases in brain edema (79.0±0.2%; 9.4±0.8µm(2)). CCI significantly up-regulated GFAP, V1aR and AQP4 protein levels and SR49059 suppressed injury induced up regulation (n=6/group). In CCI-vehicle, sham and CCI-SR49059 groups, GFAP was 1.58±0.04, 0.47±0.02, and 0.81±0.03, respectively; V1aR was 1.00±0.06, 0.45±0.05, and 0.46±0.09; and AQP4 was 2.03±0.34, 0.49±0.04, and 0.92±0.22. Confocal immunohistochemistry gave analogous results. In CCI-vehicle, sham and CCI-SR49059 groups, fluorescence intensity of GFAP was 349±38, 56±5, and 244±30, respectively, V1aR was 601±71, 117.8±14, and 390±76, and AQP4 was 818±117, 158±5, and 458±55 (n=3/group). The results support that edema was predominantly cellular following CCI and documented that V1aR inhibition with SR49059 suppressed injury-induced up regulation of GFAP, V1A and AQP4, blunting edematous changes. Our findings suggest V1aR inhibitors may be potential therapeutic tools to prevent cellular swelling and provide treatment for post-traumatic brain edema.

Real-time monitoring of changes in brain extracellular sodium and potassium concentrations and intracranial pressure after selective vasopressin-1a receptor inhibition following focal traumatic brain injury in rats.

Brain swelling and increased intracranial pressure (ICP) following traumatic brain injury (TBI) contribute to poor outcome. Vasopressin-1a receptors (V1aR) and aquaporin-4 (AQP4) regulate water transport and brain edema formation, perhaps in part by modulating cation fluxes. After focal TBI, V1aR inhibitors diminish V1aR and AQP4, reduce astrocytic swelling and brain edema. We determined whether V1aR inhibition with SR49059 after lateral controlled-cortical-impact (CCI) injury affects extracellular Na(+) and K(+) concentrations ([Na(+)]e; [K(+)]e). Ion-selective Na(+) and K(+) electrodes (ISE) and an ICP probe were implanted in rat parietal cortex, and [Na(+)]e, [K(+)]e, and physiological parameters were monitored for 5 h post-CCI. Sham-vehicle-ISE, CCI-vehicle-ISE and CCI-SR49059-ISE groups were studied, and SR49059 was administered 5 min to 5 h post-injury. We found a significant injury-induced decrease in [Na(+)]e to 80.1 ± 15 and 87.9 ± 7.9 mM and increase in [K(+)]e to 20.9 ± 3.8 and 13.4 ± 3.4 mM at 5 min post-CCI in CCI-vehicle-ISE and CCI-SR49059-ISE groups, respectively (p<0.001 vs. baseline; ns between groups). Importantly, [Na(+)]e in CCI-SR49059-ISE was reduced 5-20 min post-injury and increased to baseline at 25 min, whereas recovery in CCI-vehicle-ISE required more than 1 hr, suggesting SR49059 accelerated [Na(+)]e recovery. In contrast, [K(+)]e recovery took 45 min in both groups. Further, ICP was lower in the CCI-SR49059-ISE group. Thus, selective V1aR inhibition allowed faster [Na(+)]e recovery and reduced ICP. By augmenting the [Na(+)]e recovery rate, SR49059 may reduce trauma-induced ionic imbalance, blunting cellular water influx and edema after TBI. These findings suggest SR49059 and V1aR inhibitors are potential tools for treating cellular edema post-TBI.

Brain energy depletion in a rodent model of diffuse traumatic brain injury is not prevented with administration of sodium lactate.

Lactate has been identified as an alternative fuel for the brain in situations of increased energy demand, as following a traumatic brain injury (TBI). This study investigates the effect of treatment with sodium lactate (NaLac) on the changes in brain energy state induced by a severe diffuse TBI. Rats were assigned to one of the eight groups (n=10 per group): 1-sham, normal saline; 2-TBI, normal saline; 3-TBI, hypertonic saline; 4-TBI, 100mM NaLac, 5-TBI, 500 mM NaLac; 6-TBI, 1280 mM NaLac; 7-TBI, 2000 mM NaLac and 8-TBI-500 mM NaLac+magnesium sulfate. Cerebrums were removed 6h after trauma. Metabolites representative of the energy state (ATP, ATP-catabolites), N-acetylaspartate (NAA), antioxidant defenses (ascorbic acid, glutathione), markers of oxidative stress (malondialdehyde, ADP-ribose) and nicotinic coenzymes (NAD(+)) were measured by HPLC. TBI induced a marked decrease in the cerebral levels of ATP, NAA, ascorbic acid, glutathione and NAD(+) and a significant rise in the content of ATP-catabolites, malondialdehyde and ADP-ribose. These alterations were not ameliorated with NaLac infusion. We observed a significant reduction in cerebral NAD(+), an essential co-enzyme for mitochondrial lactate-dehydrogenase that converts lactate into pyruvate and thus replenishes the tricarboxylic acid cycle. These results suggest that the metabolic pathway necessary to consume lactate may be compromised following a severe diffuse TBI in rats.

Effect of secondary insults upon aquaporin-4 water channels following experimental cortical contusion in rats.

Although secondary insults of hypoxia and hypotension (HH) are generally considered to cause fulminant brain edema in traumatic brain injury (TBI), the combined effect of TBI with HH on brain edema and specifically the expression of aquaporin-4 (AQP4) have not been fully elucidated. The goal of this study was to document the effect of secondary insults on brain water, AQP4 expression, electrolytes, and blood-brain barrier (BBB) permeability during the acute stage of edema development. We measured brain water content and electrolytes (series 1); BBB permeability based on Evans blue (EB) dye extravasation (series 2); and AQP4 expression using immunoblotting (series 3) at 1 h and 5 h following cortical contusion injury (CCI). Secondary insults significantly worsened BBB function at 5 h post injury. Moreover, a significant reduction of upregulation on AQP4 expression was observed in trauma, coupled with a mild secondary insult of hypoxia hypotension. These findings indicate that a secondary insult following CCI at 5 h post injury worsens brain edema, disrupts ionic homeostasis, and blunts the normal upregulation of AQP4 that occurs after trauma, suggesting that the blunting of AQP4 may contribute to the detrimental effects of secondary insults.

The protein kinase C activator phorbol myristate acetate decreases brain edema by aquaporin 4 downregulation after middle cerebral artery occlusion in the rat.

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) is known to interact with aquaporin 4 (AQP 4), a water-selective transporting protein that is abundant in astrocytes, and has experimentally been found to decrease osmotically-induced cell swelling. The purpose of this study was to examine whether PMA reduces brain edema following focal ischemia induced by middle cerebral artery (MCA) occlusion by modulation of AQP4 expression. Male Sprague-Dawley rats were randomly assigned to either sham surgery (n = 6), or a continuous intravenous infusion of vehicle (1% dimethylsulfoxide), followed by MCA occlusion (n = 18), and administration of PMA at 50 microg/kg (n = 6) or at 200 microg/kg (n = 6) starting 60 min before or 30 min (200 microg/kg; n = 6) or 60 min (200 microg/kg; n = 6) after MCA occlusion. Cerebral blood flow was monitored with laser Doppler over the MCA territory, and confirmed a 70% reduction during occlusion. After a 2-h period of ischemia and 2 h of reperfusion, the animals were sacrificed for assessment of brain water content and sodium and potassium concentration. AQP4 expression was assessed by immunoblotting and quantified by densitometry (n = 24). Statistical analysis was performed by ANOVA followed by Tukey's post-hoc test. PMA treatment at 200 microg/kg significantly reduced brain water concentration in the infarcted area when started 60 min before or 30 min after occlusion (p < 0.001 and p = 0.022, respectively), and prevented the subsequent sodium shift (p < 0.05). PMA normalized the AQP4 upregulation in ischemia (p = 0.021). A downregulation of AQP4 in the ischemic area paralleling the reduction in brain edema formation following PMA treatment suggests that the effect was mediated by AQP4 modulation.

Modulation of AQP4 expression by the selective V1a receptor antagonist, SR49059, decreases trauma-induced brain edema.

BACKGROUND: Currently, there are no pharmacological treatments available for traumatically induced brain edema and the subsequent rise of ICP. Evidence indicates that Aquaporin-4 (AQP4) plays a significant role in the pathophysiology of brain edema. Previously we have reported that SR49059 reduced brain edema secondary to ischemia. We, therefore, examined whether the selective V1a receptor antagonist, SR49059, reduces brain edema by modulating AQP4 expression following cortical contusion injury (CCI). METHODS: Traumatic brain injury (TBI) was produced in thirty-two adult male Sprague-Dawley rats by lateral CCI (6.0 m/sec, 3 mm depth). Animals were randomly assigned to vehicle (n=16) or SR49059 treatment (n=16) groups and administered drug (960 microl/hr i.v.) immediately after injury over a 5 hr period. Animals were sacrificed for assessment of brain water content by Wet/Dry method and AQP4 protein expression by immunoblotting expressed as the ratio of AQP4 and Cyclophilin-A densitometries. FINDINGS: Elevated AQP4 expression levels and water content were observed on the right injured side in both the right anterior (RA) and right posterior (RP) section compared to the left non-injured side inclusive of the left anterior (LA) and right anterior (RA) sections. The average AQP4 expression levels in contused areas for animals receiving SR drug treatment (RA: 1.313 +/- 0.172, RP: 1.308 +/- 0.175) were significantly decreased from vehicle-treated animals (RA: 2.181 +/- 0.232, RP: 2.303 +/- 0.370, p = 0.001, p= 0.003). Water content levels on SR treatment (78.89 +/- 0.14) was also significantly decreased from vehicle levels (80.38 +/- 0.38, p < 0.01) in the traumatized hemisphere. CONCLUSIONS: SR49059 significantly reduced trauma-induced AQP4 up-regulation in the contused hemisphere. Moreover, brain water content was also significantly reduced paralleling the AQP4 suppression. These data provide further support that vasopressin (AVP) and V1a receptors can control water flux through astrocytic plasma membranes by regulating AQP4 expression. Taken in concert, these results affirm our laboratories contention that AQP4 can be effectively modulated pharmacologically.

The modulation of aquaporin-4 by using PKC-activator (phorbol myristate acetate) and V1a receptor antagonist (SR49059) following middle cerebral artery occlusion/reperfusion in the rat.

BACKGROUND: We have pursued the concept that traumatic brain edema is predominantly cellular and that water entry is modulated in part by aquaporins. Aquaporin-4 (AQP4) has been shown to play a significant role in cellular edema formation. Phorbol myristate acetate (PMA) is a potent PKC activator; purportedly involved in modulation of AQP4 activity. Alternatively, AQP4 may be regulated by arginine-vasopressin. Administration of the vasopressin antagonist (SR49059) reduced brain water content and sodium shift following MCAo. To investigate if edema formation is affected by the reduction of AQP4 expression, we utilized PMA and SR49059 following middle cerebral artery occlusion model (MCAo), and measured AQP4 expression by Western-Blot (WB) techniques. METHODS: Male Sprague Dawley rats were randomly assigned to sham (n=4) or MCAo groups (vehicle, PMA or SR49059 infusion; n=6 each). Each solution was infused for 5 hours, starting 1 hour before injury. After a two-hour period of ischemia and two-hour reperfusion, animals were sacrificed and brain regions of interest were processed by WB to quantify the effect of treatment on AQP4 expression. RESULTS: These studies demonstrate that MCAo results in a significant up-regulation of AQP4 on the ischemic zone when compared to the contralateral un-injured hemisphere (p < 0.05) and that PMA and SR49059 treatment significantly down-regulated AQP4 expression compared to the vehicle group (p < 0.05). CONCLUSIONS: These studies support the hypotheses that PMA and SR49059 may be useful in reducing cerebral water accumulation by modulating AQP4 expression and that pharmacological manipulation of AQP4 may emerge as a viable strategy for the reduction of fulminating edema following ischemic injury.

Administration of the immunophilin ligand FK506 differentially attenuates neurofilament compaction and impaired axonal transport in injured axons following diffuse traumatic brain injury.

Traumatic axonal injury (TAI) following traumatic brain injury (TBI) remains a clinical problem for which no effective treatment exists. TAI was thought to involve intraaxonal changes that universally led to impaired axonal transport (IAT), disconnection and axonal bulb formation. However, recent, immunocytochemical studies employing antibodies to amyloid precursor protein (APP), a marker of IAT and antibodies to neurofilament compaction (NFC), RM014, demonstrated that NFC typically occurs independent of IAT, indicating the existence of different populations of damaged axons. FK506 administration has been shown to attenuate IAT. However, in light of the above, the ability of FK506 to attenuate axonal damage demonstrating NFC requires evaluation. The current study explored the potential of FK506 to attenuate both populations of damaged axons. Rats were administered FK506 (3 mg/kg) or vehicle 30 min preinjury. Three hours post-TBI, tissue was prepared for the visualization of TAI using antibodies targeting IAT (APP) or NFC (RMO14) or a combined labeling strategy. Confirming previous reports, FK506 treatment reduced the number of axons demonstrating IAT in the CSpT, from 411 +/- 54.70 to 91.00 +/- 33.87 (P

Quantitative analysis of the relationship between intra- axonal neurofilament compaction and impaired axonal transport following diffuse traumatic brain injury.

Traumatic axonal injury (TAI) following traumatic brain injury (TBI) contributes to morbidity and mortality. TAI involves intra-axonal changes assumed to progress to impaired axonal transport (IAT), disconnection, and axonal bulb formation. Immunocytochemical studies employing antibodies to amyloid precursor protein (APP), a marker of IAT and RMO14, a marker of neurofilament compaction (NFC), have shown that TAI involves both NFC and IAT, with the suggestion that NFC leads to IAT. Recently, new data has suggested that NFC may occur independently of IAT. The objective of this study was to determine quantitatively the precise relationship between NFC and IAT. Following TBI, rats were studied at 30 min, 3 h, and 24 h. Using single-label immunocytochemistry employing the antibodies RM014, APP, or a combined labeling strategy targeting APP/RMO14 in aggregate, the immunoreactive (IR) profiles were counted in the corticospinal tract (CSpT) and medial lemniscus (ML). In the CSpT, the number of axons demonstrating RMO14-IR approximated the number of axons showing APP-IR, with the APP-IR population showing a significant increase over 24 h (p < 0.05). The sum of both single-label counts equaled the aggregate APP/RMO14 numbers, demonstrating little relationship between NFC and IAT. In the ML, 75% of fibers demonstrated a separation of APP-IR and NFC-IR; however, 25% of the ML fibers showed co-localization of APP-IR and RMO14. The results of these studies indicate that, in the majority of damaged axons, NFC is not associated with IAT. Our findings argue for the use of multiple markers when evaluating the extent of TAI or the efficacy of therapies targeting the treatment of TAI.