Vincent Vagni

Reduction of cognitive and motor deficits after traumatic brain injury in mice deficient in poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP), or poly-(ADP-ribose) synthetase, is a nuclear enzyme that consumes NAD when activated by DNA damage. The role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. Using a controlled cortical impact (CCI) model of TBI and mice deficient in PARP, the authors studied the effect of PARP on functional and histologic outcome after CCI using two protocols. In protocol 1, naïve mice (n = 7 +/+, n = 6 –/–) were evaluated for motor and memory acquisition before CCI. Mice were then subjected to severe CCI and killed at 24 hours for immunohistochemical detection of nitrated tyrosine, an indicator of peroxynitrite formation. Motor and memory performance did not differ between naïve PARP +/+ and –/– mice. Both groups showed nitrotyrosine staining in the contusion, suggesting that peroxynitrite is produced in contused brain. In protocol 2, mice (PARP +/+, n = 8; PARP –/–, n = 10) subjected to CCI were tested for motor and memory function, and contusion volume was determined by image analysis. PARP –/– mice demonstrated improved motor and memory function after CCI versus PARP +/+ mice (P < 0.05). However, contusion volume was not different between groups. The results suggest a detrimental effect of PARP on functional outcome after TBI.

Adenosine A1 receptor knockout mice develop lethal status epilepticus after experimental traumatic brain injury.

Adenosine, acting at A1 receptors, exhibits anticonvulsant effects in experimental epilepsy—and inhibits progression to status epilepticus (SE). Seizures after traumatic brain injury (TBI) may contribute to pathophysiology. Thus, we hypothesized that endogenous adenosine, acting via A1 receptors, mediates antiepileptic benefit after experimental TBI. We subjected A1-receptor knockout (ko) mice, heterozygotes, and wild-type (wt) littermates (n = 115) to controlled cortical impact (CCI). We used four outcome protocols in male mice: (1) observation for seizures, SE, and mortality in the initial 2 h, (2) assessment of seizure score (electroencephalogram (EEG)) in the initial 2 h, (3) assessment of mortality at 24 h across injury levels, and (4) serial assessment of arterial blood pressure, heart rate, blood gases, and hematocrit. Lastly, to assess the influence of gender on this observation, we observed female mice for seizures, SE, and mortality in the initial 2 h. Seizure activity was noted in 83% of male ko mice in the initial 2 h, but was seen in no heterozygotes and only 33% of wt (P < 0.05). Seizures in wt were brief (1 to 2 secs). In contrast, SE involving lethal sustained (>1 h) tonic clonic activity was uniquely seen in ko mice after CCI (50% incidence in males), (P < 0.05). Seizure score was twofold higher in ko mice after CCI versus either heterozygote or wt (P < 0.05). An injury-intensity dose–response for 24 h mortality was seen in ko mice (P < 0.05). Physiologic parameters were similar between genotypes. Seizures were seen in 100% of female ko mice after CCI versus 14% of heterozygotes and 25% wt (P < 0.05) and SE was restricted to the ko mice (83% incidence). Our data suggest a critical endogenous anticonvulsant action of adenosine at A1 receptors early after experimental TBI.

Enhanced Oxidative Stress in iNOS-Deficient Mice after Traumatic Brain Injury: Support for a Neuroprotective Role of iNOS

Studies in experimental traumatic brain injury (TBI) suggest both deleterious and protective effects of inducible nitric oxide synthase (iNOS). Early after injury, iNOS may be detrimental via formation of peroxynitrite and iNOS inhibitors are protective. In contrast, we reported impaired long-term functional outcome after TBI in iNOS knockout (ko) versus wild-type (wt) mice. To elucidate potential neuroprotective and neurotoxic mechanisms for iNOS, we studied nitric oxide formation by electron paramagnetic resonance (EPR) spectroscopy using diethyldithiocarbamate—iron (DETC—Fe) as a spin trap and markers of nitrosative (S-nitrosothiol (RSNO, Fluorescent assay); nitrotyrosine (3NT, ELISA)) and oxidative stress (ascorbate, HPLC) at 72u2009h after controlled cortical impact (CCI) in iNOS ko and wt and in uninjured iNOS ko and wt mice. 3NT immunostaining with macrophage and myeloperoxidase (MPO) dual labeling was also assessed in brain sections. Brain DETC—Fe—NO low-temperature EPR signal intensity was ∼2-fold greater in wt versus iNOS ko at 72u2009h after CCI. Ascorbate levels decreased in injured hemisphere in wt and iNOS ko versus uninjured —this decrease was more pronounced in iNOS ko. In wt mice, RSNO and 3NT levels were increased after CCI versus uninjured (50% and 400%, respectively, P<0.05). RSNO levels were not increased in iNOS ko after CCI. Nitrotyrosine levels increased after CCI in wt and ko versus respective uninjured —this increase was more pronounced in wt (2.34±0.95 versus 1.27±0.49u2009pmol/mg protein, P<0.05). Increased 3NT immunoreactivity was detected in wt versus iNOS ko at 72u2009h after CCI, and colocalized with macrophage marker and MPO. Our data support a role for iNOS-derived NO as an endogenous antioxidant after CCI. iNOS also contributes protein nitrosylation and nitration. Colocalization of 3NT with macrophages and MPO suggests generation of nitrating agents by macrophages and/or phagocytosis of nitrated proteins.

Isoflurane exerts neuroprotective actions at or near the time of severe traumatic brain injury.

Isoflurane improves outcome vs. fentanyl anesthesia, in experimental traumatic brain injury (TBI). We assessed the temporal profile of isoflurane neuroprotection and tested whether isoflurane confers benefit at the time of TBI. Adult, male rats were randomized to isoflurane (1%) or fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h) for 30 min pre-TBI. Anesthesia was discontinued, rats recovered to tail pinch, and TBI was delivered by controlled cortical impact. Immediately post-TBI, rats were randomized to 1 h of isoflurane, fentanyl, or no additional anesthesia, creating 6 anesthetic groups (isoflurane:isoflurane, isoflurane:fentanyl, isoflurane:none, fentanyl:isoflurane, fentanyl:fentanyl, fentanyl:none). Beam balance, beam walking, and Morris water maze (MWM) performances were assessed over post-trauma d1-20. Contusion volume and hippocampal survival were assessed on d21. Rats receiving isoflurane pre- and post-TBI exhibited better beam walking and MWM performances than rats treated with fentanyl pre- and any treatment post-TBI. All rats pretreated with isoflurane had better CA3 neuronal survival than rats receiving fentanyl pre- and post-TBI. In rats pretreated with fentanyl, post-traumatic isoflurane failed to affect function but improved CA3 neuronal survival vs. rats given fentanyl pre- and post-TBI. Post-traumatic isoflurane did not alter histopathological outcomes in rats pretreated with isoflurane. Rats receiving fentanyl pre- and post-TBI had the worst CA1 neuronal survival of all groups. Our data support isoflurane neuroprotection, even when used at the lowest feasible level before TBI (i.e., when discontinued with recovery to tail pinch immediately before injury). Investigators using isoflurane must consider its beneficial effects in the design and interpretation of experimental TBI research.

Hemorrhagic Shock after Experimental Traumatic Brain Injury in Mice: Effect on Neuronal Death

Traumatic brain injury (TBI) from blast injury is often complicated by hemorrhagic shock (HS) in victims of terrorist attacks. Most studies of HS after experimental TBI have focused on intracranial pressure; few have explored the effect of HS on neuronal death after TBI, and none have been done in mice. We hypothesized that neuronal death in CA1 hippocampus would be exacerbated by HS after experimental TBI. C57BL6J male mice were anesthetized with isoflurane, mean arterial blood pressure (MAP) was monitored, and controlled cortical impact (CCI) delivered to the left parietal cortex followed by continued anesthesia (CCI-only), or either 60 or 90 min of volume-controlled HS. Parallel 60- or 90-min HS-only groups were also studied. After HS (+/-CCI), 6% hetastarch was used targeting MAP of > or =50 mm Hg during a 30-min Pre-Hospital resuscitation phase. Then, shed blood was re-infused, and hetastarch was given targeting MAP of > or =60 mm Hg during a 30-min Definitive Care phase. Neurological injury was evaluated at 24 h (fluorojade C) or 7 days (CA1 and CA3 hippocampal neuron counts). HS reduced MAP to 30-40 mm Hg in all groups, p < 0.05 versus CCI-only. Ipsilateral CA1 neuron counts in the 90-min CCI+HS group were reduced at 16.5 +/- 14.1 versus 30.8 +/- 6.8, 32.3 +/- 7.6, 30.6 +/- 2.2, 28.1 +/- 2.2 neurons/100 mum in CCI-only, 60-min HS-only, 90-min HS-only, and 60-min CCI+HS, respectively, all p < 0.05. CA3 neuron counts did not differ between groups. Fluorojade C staining confirmed neurodegeneration in CA1 in the 90-min CCI+HS group. Our data suggest a critical time window for exacerbation of neuronal death by HS after CCI and may have implications for blast injury victims in austere environments where definitive management is delayed.

Adenosine A1 receptor activation as a brake on the microglial response after experimental traumatic brain injury in mice.

We reported that adenosine A(1) receptor (A(1)AR) knockout (KO) mice develop lethal status epilepticus after experimental traumatic brain injury (TBI), which is not seen in wild-type (WT) mice. Studies in epilepsy, multiple sclerosis, and neuro-oncology suggest enhanced neuro-inflammation and/or neuronal death in A(1)AR KO. We hypothesized that A(1)AR deficiency exacerbates the microglial response and neuronal damage after TBI. A(1)AR KO and WT littermates were subjected to mild controlled cortical impact (3 m/sec; 0.5 mm depth) to left parietal cortex, an injury level below the acute seizure threshold in the KO. At 24 h or 7 days, mice were sacrificed and serial sections prepared. Iba-1 immunostaining was used to quantify microglia at 7 days. To assess neuronal injury, sections were stained with Fluoro-Jade C (FJC) at 24 h to evaluate neuronal death in the hippocampus and cresyl violet staining at 7 days to analyze cortical lesion volumes. We also studied the effects of adenosine receptor agonists and antagonists on (3)H-thymidine uptake (proliferation index) by BV-2 cells (immortalized mouse microglial). There was no neuronal death in CA1 or CA3 quantified by FJC. A(1)AR KO mice exhibited enhanced microglial response; specifically, Iba-1 + microglia were increased 20-50% more in A(1)AR KO versus WT in ipsilateral cortex, CA3, and thalamus, and contralateral cortex, CA1, and thalamus (p < 0.05). However, contusion and cortical volumes did not differ between KO and WT. Pharmacological studies in cultured BV-2 cells indicated that A(1)AR activation inhibits microglial proliferation. A(1)AR activation is an endogenous inhibitor of the microglial response to TBI, likely via inhibition of proliferation, and this may represent a therapeutic avenue to modulate microglia after TBI.

Disruption of Bax Protein Prevents Neuronal Cell Death but Produces Cognitive Impairment in Mice following Traumatic Brain Injury

Apoptosis contributes to delayed neuronal cell death in traumatic brain injury (TBI). To investigate if Bax plays a role in neuronal cell death and functional outcome after TBI, Bax gene disrupted (null) mice and wild-type (WT) controls were subjected to the controlled cortical impact (CCI) model of TBI. Motor function in WT and Bax null mice was evaluated using the round beam balance and the wire grip test on days 0-5. Spatial memory was assessed using a Morris Water Maze adopted for mice on days 14-18 post-injury. For histopathological analysis, animals were sacrificed 24 h and 21 days post-injury. In all three behavioral tests, the sham and TBI-injured Bax null mice performed significantly worse than their WT sham and TBI-injured counterparts. However, Bax null mice exhibited a higher percentage of surviving neurons in the CA1 and CA3 regions of hippocampus measured at 21 days post-injury. At 24 h after trauma, Bax null mice had fewer TUNEL positive cells in the CA1 and dentate regions of hippocampus as compared to WT mice, suggesting that deletion of the Bax gene ameliorates hippocampal cell death after TBI. Sham-operated Bax null mice had significantly greater brain volume as compared to WT mice. Thus, it is possible that Bax deficiency in the transgenic mice produces developmental behavioral effects, perhaps due to Baxs role in regulating cell death during development.

Resuscitation of Traumatic Brain Injury and Hemorrhagic Shock with Polynitroxylated Albumin, Hextend, Hypertonic Saline, and Lactated Ringer's: Effects on Acute Hemodynamics, Survival, and Neuronal Death in Mice

Outcome after traumatic brain injury (TBI) is worsened by hemorrhagic shock (HS), but the optimal resuscitation approach is unclear. In particular, treatment of TBI patients with colloids remains controversial. We hypothesized that resuscitation with the colloids polynitroxylated albumin (PNA) or Hextend (HEX) is equal or superior to resuscitation with the crystalloids hypertonic (3%) saline (HTS) or lactated Ringers solution (LR) after TBI plus HS in mice. C57/BL6 mice (n = 30) underwent controlled cortical impact (CCI) and 90 min of volume-controlled HS (2 mL/100 g). The mice were randomized to resuscitation with LR, HEX, HTS, or PNA, followed by 30 min of test fluid administration targeting a mean arterial pressure (MAP) of >50 mm Hg. Shed blood was re-infused to target a MAP >70 mm Hg. At 7 days post-insult, hippocampal neuron counts were assessed in hematoxylin and eosin-stained sections to quantify neuronal damage. Prehospital MAP was higher, and prehospital and total fluid requirements were lower in the PNA and HEX groups (p < 0.05 versus HTS or LR). Also, 7-day survival was highest in the PNA group, but was not significantly different than the other groups. Ipsilateral hippocampal CA1 and CA3 neuron loss did not differ between groups. We conclude that the colloids PNA and HEX exhibited more favorable effects on acute resuscitation parameters than HTS or LR, and did not increase hippocampal neuronal death in this model.

Polynitroxylated pegylated hemoglobin: A novel neuroprotective hemoglobin for acute volume-limited fluid resuscitation after combined traumatic brain injury and hemorrhagic hypotension in mice

Objective:Resuscitation of hemorrhagic hypotension after traumatic brain injury is challenging. A hemoglobin-based oxygen carrier may offer advantages. The novel therapeutic hemoglobin-based oxygen carrier, polynitroxylated pegylated hemoglobin (PNPH), may represent a neuroprotective hemoglobin-based oxygen carrier for traumatic brain injury resuscitation. Hypotheses:1) PNPH is a unique non-neurotoxic hemoglobin-based oxygen carrier in neuronal culture and is neuroprotective in in vitro neuronal injury models. 2) Resuscitation with PNPH would require less volume to restore mean arterial blood pressure than lactated Ringers or Hextend and confer neuroprotection in a mouse model of traumatic brain injury plus hemorrhagic hypotension. Design:Prospective randomized, controlled experimental study. Setting:University center. Measurements and Main Results:In rat primary cortical neuron cultures, control bovine hemoglobin was neurotoxic (lactate dehydrogenase release; 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide assay) at concentrations from 12.5 to 0.625 &mgr;M, whereas polyethylene glycol-conjugated hemoglobin showed intermediate toxicity. PNPH was not neurotoxic (p < .05 vs. bovine hemoglobin and polyethylene glycol hemoglobin; all concentrations). PNPH conferred neuroprotection in in vitro neuronal injury (glutamate/glycine exposure and neuronal stretch), as assessed via lactate dehydrogenase and 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide (all p < .05 vs. control). C57BL6 mice received controlled cortical impact followed by hemorrhagic hypotension (2 mL/100 g, mean arterial blood pressure ∼35–40 mm Hg) for 90 min. Mice were resuscitated (mean arterial blood pressure >50 mm Hg for 30 min) with lactated Ringers, Hextend, or PNPH, and then shed blood was reinfused. Mean arterial blood pressures, resuscitation volumes, blood gasses, glucose, and lactate were recorded. Brain sections at 7 days were examined via hematoxylin and eosin and Fluoro-Jade C (identifying dying neurons) staining in CA1 and CA3 hippocampus. Resuscitation with PNPH or Hextend required less volume than lactated Ringers (both p < .05). PNPH but not Hextend improved mean arterial blood pressure vs. lactated Ringers (p < .05). Mice resuscitated with PNPH had fewer Fluoro-Jade C positive neurons in CA1 vs. Hextend and lactated Ringers, and CA3 vs. Hextend (p < .05). Conclusions:PNPH is a novel neuroprotective hemoglobin-based oxygen carrier in vitro and in vivo that may offer unique advantages for traumatic brain injury resuscitation.

Administration of adenosine receptor agonists or antagonists after controlled cortical impact in mice: effects on function and histopathology

Adenosine is an endogenous neuroprotectant via anti-excitotoxic effects at A(1) receptors, and blood flow promoting and anti-inflammatory effects at A(2a) receptors. Previous studies showed improved motor function after fluid percussion injury (FPI) in rats treated with the broad-spectrum adenosine receptor agonist 2-chloroadenosine (2-CA). We studied the effects of 2-CA, a specific A(1) agonist (2-chloro-N(6)-cyclopentyladenosine, CCPA), and a specific A(1) antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX) on motor task and Morris water maze (MWM) performance, and histopathology (contusion volume, hippocampal cell counts) after controlled cortical impact (CCI) in mice. Each agent (12 nmol), or respective vehicle (saline or DMSO) was injected into dorsal hippocampus beneath the contusion immediately after CCI or craniotomy (sham). 2-CA treatment attenuated wire grip deficits after CCI (P<0.05 versus other treatments). DPCPX treatment exacerbated deficits on beam balance (P<0.05 versus sham). No treatment effect was seen on MWM performance, although there was a deleterious effect of the DMSO vehicle used for DPCPX. Contusion volume tended to be attenuated by 2-CA (P=0.08 versus saline) and increased after either DMSO or DPCPX (P<0.05 versus all groups). CA1 and CA3 counts were decreased in all groups versus sham. However, treatment with the selective A(1) agonist CCPA attenuated the CA3 cell loss (P<0.05 versus other treatment). We suggest that the beneficial effect of the broad spectrum adenosine receptor agonist 2-CA on motor function after CCI is not mediated solely by effects at the A(1) receptor.