Jimmy Zhang

Clinical correlates in an experimental model of repetitive mild brain injury.

Although there is growing awareness of the long‐term cognitive effects of repetitive mild traumatic brain injury (rmTBI; eg, sports concussions), whether repeated concussions cause long‐term cognitive deficits remains controversial. Moreover, whether cognitive deficits depend on increased amyloid β deposition and tau phosphorylation or are worsened by the apolipoprotein E4 allele remains unknown. Here, we use an experimental model of rmTBI to address these clinical controversies.

Low-Level Laser Light Therapy Improves Cognitive Deficits and Inhibits Microglial Activation after Controlled Cortical Impact in Mice

Low-level laser light therapy (LLLT) exerts beneficial effects on motor and histopathological outcomes after experimental traumatic brain injury (TBI), and coherent near-infrared light has been reported to improve cognitive function in patients with chronic TBI. However, the effects of LLLT on cognitive recovery in experimental TBI are unknown. We hypothesized that LLLT administered after controlled cortical impact (CCI) would improve post-injury Morris water maze (MWM) performance. Low-level laser light (800 nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60-80 min after CCI. Injured mice treated with 60 J/cm² (500 mW/cm²×2 min) either transcranially or via an open craniotomy had modestly improved latency to the hidden platform (p<0.05 for group), and probe trial performance (p<0.01) compared to non-treated controls. The beneficial effects of LLLT in open craniotomy mice were associated with reduced microgliosis at 48 h (21.8±2.3 versus 39.2±4.2 IbA-1+ cells/200×field, p<0.05). Little or no effect of LLLT on post-injury cognitive function was observed using the other doses, a 4-h administration time point and 7-day administration of 60 J/cm². No effect of LLLT (60 J/cm² open craniotomy) was observed on post-injury motor function (days 1-7), brain edema (24 h), nitrosative stress (24 h), or lesion volume (14 days). Although further dose optimization and mechanism studies are needed, the data suggest that LLLT might be a therapeutic option to improve cognitive recovery and limit inflammation after TBI.

Combination therapy targeting Akt and mammalian target of rapamycin improves functional outcome after controlled cortical impact in mice

Akt and mammalian target of rapamycin (mTOR) are both activated after traumatic brain injury (TBI), however complex interplay between the two hampers deciphering their functional implications in vivo. We examined the effects of single and combination inhibitors of Akt/mTOR in a mouse controlled cortical impact (CCI) model. Following CCI, phospho-Akt-473 (p-Akt) and -S6 ribosomal protein (p-S6RP), a downstream substrate of mTOR, were increased in cortical and hippocampal brain homogenates (P<0.05 versus sham). At 24 hours, p-S6RP was detected in neurons and was robustly induced in microglia and astrocytes in injured hippocampus. In vivo activity of Akt and mTOR inhibitors administered separately was confirmed by reduced expression of p-GSK3β (P<0.01) or p-S6RP (P<0.05), respectively, after CCI. Importantly, administration of Akt and mTOR inhibitors together (but not of either alone) improved postinjury motor (P=0.02) and cognitive deficits (hidden platform trials, P=0.001; probe trials, P<0.05), decreased propidium iodide-positive cells in CA1 and CA3 (P<0.005), and unexpectedly increased p-GSK3β in hippocampus. Although the roles of Akt and mTOR in the pathogenesis of TBI remain to be fully elucidated, dual inhibition of Akt and mTOR may have therapeutic potential for TBI.

Tumor necrosis factor alpha and Fas receptor contribute to cognitive deficits independent of cell death after concussive traumatic brain injury in mice

Tumor necrosis factor alpha (TNFα) and Fas receptor contribute to cell death and cognitive dysfunction after focal traumatic brain injury (TBI). We examined the role of TNFα/Fas in postinjury functional outcome independent of cell death in a novel closed head injury (CHI) model produced with weight drop and free rotational head movement in the anterior–posterior plane. The CHI produced no cerebral edema or blood–brain barrier damage at 24 to 48 hours, no detectable cell death, occasional axonal injury (24 hours), and no brain atrophy or hippocampal cell loss (day 60). Microglia and astrocytes were activated (48 to 72 hours). Tumor necrosis factor-α mRNA, Fas mRNA, and TNFα protein were increased in the brain at 3 to 6 hours after injury (P < 0.001 versus sham injured). In wild-type (WT) mice, CHI produced hidden platform (P = 0.009) and probe deficits (P = 0.001) in the Morris water maze versus sham. Surprisingly, injured TNFα/Fas knockout (KO) mice performed worse in hidden platform trials (P = 0.036) but better in probe trials than did WT mice (P = 0.0001). Administration of recombinant TNFα to injured TNFα/Fas KO mice reduced probe trial performance to that of WT. Thus, TNFα/Fas influence cognitive deficits independent of cell death after CHI. Therapies targeting TNFα/Fas together may be inappropriate for patients with concussive TBI.

Age-Dependent Effect of Apolipoprotein E4 on Functional Outcome after Controlled Cortical Impact in Mice:

The apolipoprotein E4 (APOE4) gene leads to increased brain amyloid beta (Aβ) and poor outcome in adults with traumatic brain injury (TBI); however, its role in childhood TBI is controversial. We hypothesized that the transgenic expression of human APOE4 worsens the outcome after controlled cortical impact (CCI) in adult but not immature mice. Adult and immature APOE4 mice had worse motor outcome after CCI (P<0.001 versus wild type (WT)), but the Morris water maze performance was worse only in adult APOE4 mice (P=0.028 at 2 weeks, P=0.019 at 6 months versus WT), because immature APOE4 mice had performance similar to WT for up to 1 year after injury. Brain lesion size was similar in adult APOE4 mice but was decreased (P=0.029 versus WT) in injured immature APOE4 mice. Microgliosis was similar in all groups. Soluble brain Aβ40 was increased at 48 hours after CCI in adult and immature APOE4 mice and in adult WT (P<0.05), and was dynamically regulated during the chronic period by APOE4 in adults but not immature mice. The data suggest age-dependent effects of APOE4 on cognitive outcome after TBI, and that therapies targeting APOE4 may be more effective in adults versus children with TBI.

Kollidon VA64, a membrane-resealing agent, reduces histopathology and improves functional outcome after controlled cortical impact in mice

Loss of plasma membrane integrity is a feature of acute cellular injury/death in vitro and in vivo. Plasmalemma-resealing agents are protective in acute central nervous system injury models, but their ability to reseal cell membranes in vivo has not been reported. Using a mouse controlled cortical impact (CCI) model, we found that propidium iodide-positive (PI+) cells pulse labeled at 6, 24, or 48 hours maintained a degenerative phenotype and disappeared from the injured brain by 7 days, suggesting that plasmalemma permeability is a biomarker of fatal cellular injury after CCI. Intravenous or intracerebroventricular administration of Kollidon VA64, poloxamer P188, or polyethylene glycol 8000 resealed injured cell membranes in vivo (P<0.05 versus vehicle or poloxamer P407). Kollidon VA64 (1 mmol/L, 500 μL) administered intravenously to mice 1 hour after CCI significantly reduced acute cellular degeneration, chronic brain tissue damage, brain edema, blood—brain barrier damage, and postinjury motor deficits (all P<0.05 versus vehicle). However, VA64 did not rescue pulse-labeled PI+ cells from eventual demise. We conclude that PI permeability within 48 hours of CCI is a biomarker of eventual cell death/loss. Kollidon VA64 reduces secondary damage after CCI by mechanisms other than or in addition to resealing permeable cells.

Detrimental effect of genetic inhibition of B-site APP-cleaving enzyme 1 on functional outcome after controlled cortical impact in young adult mice.

β-Amyloid (Aβ) peptides, most notably associated with Alzheimers disease, have been implicated in the pathogenesis of secondary injury after traumatic brain injury (TBI). A prior study has demonstrated that blocking the β-site amyloid precursor protein (APP)-cleaving enzyme 1 (Bace1) required for production of Aβ from APP improved functional and histologic outcomes after controlled cortical impact (CCI) in aged mice. However, the majority of patients with severe TBI are young adults under the age of 40. Prior experimental models have suggested age-dependent differences in Aβ clearance, and a recent study in our lab suggests that young animals remediate acute elevations in Aβ after CCI better than older animals. We therefore tested the hypothesis that Bace1 deletion in young adult mice would not be protective after CCI. Male Bace1 knockout (Bace1(-/-)) and wild-type Bace1(+/+) (C57BL/6) mice (2-3 months old) were subjected to CCI (n=18-23/group) or sham injury (n=10-12/group). Functional outcomes were assessed with wire grip (motor) and the Morris water maze (MWM; spatial memory). Soluble Aβ levels were assessed at 48 h after CCI. Histopathological outcomes were assessed by lesion and hippocampal volume. Clustered ordinal logistic regression showed overall significant impairment in motor performance in injured Bace1(-/-) versus Bace1(+/+) animals (p=0.003). No significant differences in MWM performance were found on repeated-measures ANOVA (p=0.11) between groups. Probe scores were significantly worse in injured Bace1(-/-) versus Bace1(+/+) mice (p=0.0009). Soluble Aβ(40) was significantly lower in ipsilateral hemispheres of Bace1(-/-) than in Bace1(+/+) animals after CCI (0.9 [IQR 0.88-0.94] pmol/g protein versus 3.8 [IQR 2.4-6.0] pmol/g protein; p=0.005). Lesion and hippocampal volumes did not differ between injured groups. The data suggest that therapies targeting Bace1 may need to be tailored according to age and injury severity, as their use may exacerbate functional deficits after TBI in younger or less severely injured patients.

Concussive injury before or after controlled cortical impact exacerbates histopathology and functional outcome in a mixed traumatic brain injury model in mice.

Traumatic brain injury (TBI) may involve diverse injury mechanisms (e.g., focal impact vs. diffuse impact loading). Putative therapies developed in TBI models featuring a single injury mechanism may fail in clinical trials if the model does not fully replicate multiple injury subtypes, which may occur concomitantly in a given patient. We report development and characterization of a mixed contusion/concussion TBI model in mice using controlled cortical impact (CCI; 0.6 mm depth, 6 m/sec) and a closed head injury (CHI) model at one of two levels of injury (53 vs. 83 g weight drop from 66 in). Compared with CCI or CHI alone, sequential CCI-CHI produced additive effects on loss of consciousness (p<0.001), acute cell death (p<0.05), and 12-day lesion size (p<0.05) but not brain edema or 48-h contusion volume. Additive effects of CHI and CCI on post-injury motor (p<0.05) and cognitive (p<0.005) impairment were observed with sequential CCI-CHI (83 g). The data suggest that concussive forces, which in isolation do not induce histopathological damage, exacerbate histopathology and functional outcome after cerebral contusion. Sequential CHI-CCI may model complex injury mechanisms that occur in some patients with TBI and may prove useful for testing putative therapies.

The pharmacokinetics and pharmacodynamics of Kollidon VA64 dissociate its protective effects from membrane resealing after controlled cortical impact in mice

Membrane-resealing agents such as poloxamer P188 improve the outcome in experimental brain injury paradigms; however, whether membrane resealing is a key mechanism for protection has not been shown in vivo. We previously reported that Kollidon VA64, a polymeric membrane-resealing agent, reduces cell membrane permeability and improves brain edema, brain tissue damage, and functional outcome after controlled cortical impact in mice, without rescuing resealed cells from death. To reconcile these disparate findings, we used a dual-pulse labeling protocol to determine membrane-resealing kinetics by VA64/P188 in vivo. Membrane resealing after controlled cortical impact in mice by intravenous or intracerebroventricular VA64 and poloxamer P188 was transient, with most cells becoming repermeabilized within 2 hours, even with multiple-dose paradigms that maintained high VA64 blood levels. Moreover, VA64 reduced cytotoxic brain edema in a water intoxication model devoid of plasmalemma permeability (P<0.05 versus P188, VA30, mannitol, and vehicle). We conclude that VA64 reduces cytotoxic and traumatic brain edema independent of membrane resealing. The results suggest that classic membrane-resealing agents such as poloxamer P188, and the newly discovered VA64, exert protective effects in central nervous system injury paradigms by mechanisms other than or in addition to maintaining permeable cell membranes sealed.

Beneficial effect of amyloid beta after controlled cortical impact

Background: Worse functional outcomes after controlled cortical impact (CCI) in Bace1−/− mice have previously been demonstrated. This study investigated whether reconstitution of amyloid-beta (Aβ) after CCI in Bace1−/− animals would reverse the detrimental effect of Bace1 deletion. Methods: Bace1−/− and wild type Bace1+/+ (C57Bl/6) mice were subjected to CCI (n = 14–23/group) or sham injury (n = 6/group). After injury, mice underwent intracerebroventricular injections of Aβ40 (n = 23 Bace1−/− and 17 Bace1+/+ per group) or vehicle (n = 14 Bace1−/− and 22 Bace1+/+ per group). Functional outcomes were assessed with wire grip (motor) and Morris water maze (spatial memory). Soluble Aβ levels were assessed at 24 hours and 21 days after CCI. Lesion volume was assessed 21 days after injury. Results: At 24 hours after injury, Aβ-treated Bace1−/− mice had Aβ40 levels similar to vehicle-treated Bace1+/+ mice, but by 21 days after injury there were no differences between Aβ-treated versus vehicle-treated Bace1−/− mice. Reconstitution with Aβ40 improved motor but not spatial memory or histopathological outcome in injured Bace1−/− mice. In contrast, treatment with Aβ40 worsened motor performance in Bace1+/+ mice. Conclusions: The data suggest Aβ40 may have some beneficial effects after CCI in young adult mice and that therapies targeting BACE should be approached cautiously.