CCR2 deficiency alters activation of microglia subsets in traumatic brain injury.

In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2-/- mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2-/- TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets.

Effects of Veliparib on Microglial Activation and Functional Outcomes after Traumatic Brain Injury in the Rat and Pig.

The inflammation response induced by brain trauma can impair recovery. This response requires several hours to develop fully and thus provides a clinically relevant therapeutic window of opportunity. Poly(ADP-ribose) polymerase inhibitors suppress inflammatory responses, including brain microglial activation. We evaluated delayed treatment with veliparib, a poly(ADP-ribose) polymerase inhibitor, currently in clinical trials as a cancer therapeutic, in rats and pigs subjected to controlled cortical impact (CCI). In rats, CCI induced a robust inflammatory response at the lesion margins, scattered cell death in the dentate gyrus, and a delayed, progressive loss of corpus callosum axons. Pre-determined measures of cognitive and motor function showed evidence of attentional deficits that resolved after three weeks and motor deficits that recovered only partially over eight weeks. Veliparib was administered beginning 2 or 24 h after CCI and continued for up to 12 days. Veliparib suppressed CCI-induced microglial activation at doses of 3 mg/kg or higher and reduced reactive astrocytosis and cell death in the dentate gyrus, but had no significant effect on delayed axonal loss or functional recovery. In pigs, CCI similarly induced a perilesional microglial activation that was attenuated by veliparib. CCI in the pig did not, however, induce detectable persisting cognitive or motor impairment. Our results showed veliparib suppression of CCI-induced microglial activation with a delay-to-treatment interval of at least 24 h in both rats and pigs, but with no associated functional improvement. The lack of improvement in long-term recovery underscores the complexities in translating anti-inflammatory effects to clinically relevant outcomes.

Radially branched deployment for more efficient cell transplantation at the scale of the human brain.

BACKGROUND: In preclinical studies, cell transplantation into the brain has shown great promise for the treatment of a wide range of neurological diseases. However, the use of a straight cannula and syringe for cell delivery to the human brain does not approximate cell distribution achieved in animal studies. This technical deficiency may limit the successful clinical translation of cell transplantation. OBJECTIVE: To develop a stereotactic device that effectively distributes viable cells to the human brain. Our primary aims were to (1) minimize the number of transcortical penetrations required for transplantation, (2) reduce variability in cell dosing and (3) increase cell survival. METHODS: We developed a modular cannula system capable of radially branched deployment (RBD) of a cell delivery catheter at variable angles from the longitudinal device axis. We also developed an integrated catheter-plunger system, eliminating the need for a separate syringe delivery mechanism. The RBD prototype was evaluated in vitro and in vivo with subcortical injections into the swine brain. Performance was compared to a 20G straight cannula with dual side ports, a device used in current clinical trials. RESULTS: RBD enabled therapeutic delivery in a precise 'tree-like' pattern branched from a single initial trajectory, thereby facilitating delivery to a volumetrically large target region. RBD could transplant materials in a radial pattern up to 2.0 cm from the initial penetration tract. The novel integrated catheter-plunger system facilitated manual delivery of small and precise volumes of injection (1.36 ± 0.13 µl per cm of plunger travel). Both dilute and highly concentrated neural precursor cell populations tolerated transit through the device with high viability and unaffected developmental potential. While reflux of infusate along the penetration tract was problematic with the use of the 20G cannula, RBD was resistant to this source of cell dose variability in agarose. RBD enabled radial injections to the swine brain when used with a modern clinical stereotactic system. CONCLUSIONS: By increasing the total delivery volume through a single transcortical penetration in agarose models, RBD strategy may provide a new approach for cell transplantation to the human brain. Incorporation of RBD or selected aspects of its design into future clinical trials may increase the likelihood of successful translation of cell-based therapy to the human patient.

Post-injury conditioning with lipopolysaccharide or lipooligosaccharide reduces inflammation in the brain.

BACKGROUND: Traumatic brain injury (TBI) is a leading cause of mortality and disability in the Western world. The first stage of TBI results from the mechanical damage from an impact or blast. A second stage occurs as an inflammatory response to the primary injury and presents an opportunity for clinical intervention. In this study, we investigated the effect of pre- and post-injury treatment with lipopolysaccharide (LPS) from Escherichia coli and lipooligosaccharide (LOS) from Neisseria meningitidis on levels of cerebral inflammatory cells, circulating blood cells, and pro- and anti-inflammatory cytokine levels in a rat model of neuroinflammation induced by intrastriatal injection of IL-1ß to mimic the second stage of TBI. METHODS: LPS or LOS was administered intravenously (IV) or intranasally (IN) 2h pre- or post-injection of IL-1ß. The rats were euthanized 12h following IL-1ß injection. Brain sections were immunostained with antibody to ED-1, a microglia cell marker. Cells in whole blood were assessed with a VetScan HM2 analyzer, and cytokine levels in sera were analyzed with a Bio-Plex system. RESULTS: Pre- and post-injury IV administration of LPS or LOS significantly reduced microglia in the brain, and IN pre-treatment with LPS or LOS showed a statistical trend towards reducing microglia. Pre- and post-treatment IV with LOS increased circulating levels of IL-2 and IL-4, whereas IN post-treatment with LPS reduced levels of the inflammatory cytokines, TNF-α and IFN-γ. CONCLUSIONS: The findings strongly support continued investigation of post-conditioning with LPS or LOS as potential neuroprotective treatments for neuroinflammation from TBI.

Intranasal delivery of deferoxamine reduces spatial memory loss in APP/PS1 mice.

Intranasal administration, which bypasses the blood-brain barrier and minimizes systemic exposure, is a non-invasive alternative for targeted drug delivery to the brain. While identification of metal dysregulation in Alzheimer's brain has led to the development of therapeutic metal-binding agents, targeting to the brain has remained an issue. The purpose of this study was to both determine concentrations of deferoxamine (DFO), a high-affinity iron chelator, reaching the brains of mice after intranasal administration and to determine its efficacy in a mouse model of spatial memory loss. Intranasal administration of DFO (2.4 mg) labeled with (59)Fe (75 µCi) to C57 mice resulted in micromolar concentrations at 30 min within brain parenchyma. After 3 months of intranasal DFO treatment, 2.4 mg three times per week, 48-week-old APP/PS1 mice had significantly reduced escape latencies in Morris water maze compared to vehicle-treated mice. This is the first report that intranasal DFO improves spatial memory in a mouse model of Alzheimer's disease and demonstrates that intranasal DFO reaches the brain in therapeutic doses.

Post-injury treatment with lipopolysaccharide or lipooligosaccharide protects rat neuronal and glial cell cultures.

Traumatic brain injury (TBI) is a major cause of disability in civilians and military personnel worldwide that is caused by the acceleration force of a primary shockwave, blast wind or the force of a direct contact. Following the primary injury, secondary injury is caused by activation of the immune response due to an influx of neuro-inflammatory cells, increased production of inflammatory cytokines, and edema. In ischemia models pre-conditioning with lipopolysaccharide (LPS) has been shown to be neuroprotective, and post-injury conditioning with LPS was found to be protective in a spinal cord and an acute brain injury model. In this study, we utilized an in vitro scratch model of TBI to assess the effect of post-injury treatment with Escherichia coli LPS and Neisseria meningitidis lipooligosaccharide (LOS) on cell death and cytokine induction by assessing the level of lactate dehydrogenase released from cells and rat multiplex cytokine assays. Our results showed that post-injury treatment of C6 glioma cells with either the LPS or the LOS reduced cell death when compared to scratched controls treated with media only. Post-injury treatment of the primary mixed neuronal cultures with LPS reduced cell death and resulted in a significant up-regulation in IL-10 when compared to controls. With LOS post-scratch treatment of the primary cell cultures, we found that IL-1α, IL-1ß, IL-6, and TNF-α were significantly upregulated in addition to IL-10 compared to the media-only controls. The results strongly support additional testing of the neuroprotective ability of post-injury treatment with LPS or LOS in models of TBI.

The effect of reductive ventricular osmotherapy on the osmolarity of artificial cerebrospinal fluid and the water content of cerebral tissue ex vivo.

The purpose of this study was to explore a novel treatment involving removal of free water from ventricular cerebrospinal fluid (CSF) for the reduction of cerebra]l edema. The hypothesis is that removal of free water from the CSF will increase the osmolarity of the CSF, which will favor movement of tissue-bound water into the ventricles, where the water can be removed. Reductive ventricular osmotherapy (RVOT) was tested in a flowing solution of artificial CSF (aCSF) with two end-points: (1) the effect of RVOT on osmolarity of the CSF, and (2) the effect of RVOT on water content of ex vivo cerebral tissue. RVOT catheters are made up of membranes permeable only to water vapor. When a sweep gas is drawn through the catheter, free water in the form of water vapor is removed from the solution. With RVOT treatment, aCSF osmolarity increased from a baseline osmolarity of 318.8 ± 0.8 mOsm/L to 339.0 ± 3.3 mOsm/L (mean ± standard deviation) within 2 h. After 10 h of treatment, aCSF osmolarity approached an asymptote at 344.0 ± 4.2 mOsm/L, which was significantly greater than control aCSF osmolarity (p <<0.001 by t-test, n = 8). Water content at the end of 6 h of circulating aCSF exposure was 6.4 ± 0.9 g H2O (g dry wt)⁻¹ in controls, compared to 6.1 ± 0.7 g H2O (g dry wt)⁻ after 6 h of RVOT treatment of aCSF (p = 0.02, n = 24). The results support the potential of RVOT as a treatment for cerebral edema and intracranial hypertension.

Intranasal deferoxamine provides increased brain exposure and significant protection in rat ischemic stroke.

Deferoxamine (DFO) is a high-affinity iron chelator approved by the Food and Drug Administration for treating iron overload. Preclinical research suggests that systemically administered DFO prevents and treats ischemic stroke damage and intracerebral hemorrhage. However, translation into human trials has been limited, probably because of difficulties with DFO administration. A noninvasive method of intranasal administration has emerged recently as a rapid way to bypass the blood-brain barrier and target therapeutic agents to the central nervous system. We report here that intranasal administration targets DFO to the brain and reduces systemic exposure, and that intranasal DFO prevents and treats stroke damage after middle cerebral artery occlusion (MCAO) in rats. A 6-mg dose of DFO resulted in significantly higher DFO concentrations in the brain (0.9-18.5 microM) at 30 min after intranasal administration than after intravenous administration (0.1-0.5 microM, p < 0.05). Relative to blood concentration, intranasal delivery increased targeting of DFO to the cortex approximately 200-fold compared with intravenous delivery. Intranasal administration of three 6-mg doses of DFO did not result in clinically significant changes in blood pressure or heart rate. Pretreatment with intranasal DFO (three 6-mg doses) 48 h before MCAO significantly decreased infarct volume by 55% versus control (p < 0.05). In addition, post-treatment with intranasal administration of DFO (six 6-mg doses) immediately after reperfusion significantly decreased infarct volume by 55% (p < 0.05). These experiments suggest that intranasally administered DFO may be a useful treatment for stroke, and a prophylactic for patients at high risk for stroke.

Use of hemoglobin-based oxygen-carrying solution-201 to improve resuscitation parameters and prevent secondary brain injury in a swine model of traumatic brain injury and hemorrhage: laboratory investigation.

OBJECT: Traumatic brain injury (TBI) often occurs as part of a multisystem trauma that may lead to hemorrhagic shock. Effective resuscitation and restoration of oxygen delivery to the brain is important in patients with TBI because hypotension and hypoxia are associated with poor outcome in head injury. We studied the effects of hemoglobin-based oxygen-carrying (HBOC)-201 solution compared with lactated Ringer (LR) solution in a large animal model of brain injury and hemorrhage, in a blinded prospective randomized study. METHODS: Swine underwent brain impact injury and hemorrhage to a mean arterial pressure (MAP) of 40 mm Hg. Twenty swine were randomized to undergo resuscitation with HBOC-201 (6 ml/kg) or LR solution (12 ml/kg) and were observed for an average of 6.5 +/- 0.5 hours following resuscitation. At the end of the observation period, magnetic resonance (MR) imaging was performed. Histological studies of swine brains were performed using Fluoro-Jade B, a marker of early neuronal degeneration. RESULTS: Swine resuscitated with HBOC-201 had higher MAP, higher cerebral perfusion pressure (CPP), improved base deficit, and higher brain tissue oxygen tension (PbtO(2)) than animals resuscitated with LR solution. No significant difference in total injury volume on T2-weighted MR imaging was observed between animals resuscitated with HBOC-201 solution (1155 +/- 374 mm(3)) or LR solution (1246 +/- 279 mm(3); p = 0.55). On the side of impact injury, no significant difference in the mean number of Fluoro-Jade B-positive cells/hpf was seen between HBOC-201 solution (61.5 +/- 14.7) and LR solution (48.9 +/- 17.7; p = 0.13). Surprisingly, on the side opposite impact injury, a significant increase in Fluoro-Jade B-positive cells/hpf was seen in animals resuscitated with LR solution (42.8 +/- 28.3) compared with those resuscitated with HBOC-201 solution (5.6 +/- 8.1; p < 0.05), implying greater neuronal injury in LR-treated swine. CONCLUSIONS: The improved MAP, CPP, and PbtO(2) observed with HBOC-201 solution in comparison with LR solution indicates that HBOC-201 solution may be a preferable agent for small-volume resuscitation in brain-injured patients with hemorrhage. The use of HBOC-201 solution appears to decrease cellular degeneration in the brain area not directly impacted by the primary injury. Hemoglobin-based oxygen-carrying-201 solution may act by improving cerebral blood flow or increasing the oxygen-carrying capacity of blood, mitigating a second insult to the injured brain.

Iron in chronic brain disorders: imaging and neurotherapeutic implications.

Iron is important for brain oxygen transport, electron transfer, neurotransmitter synthesis, and myelin production. Though iron deposition has been observed in the brain with normal aging, increased iron has also been shown in many chronic neurological disorders including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. In vitro studies have demonstrated that excessive iron can lead to free radical production, which can promote neurotoxicity. However, the link between observed iron deposition and pathological processes underlying various diseases of the brain is not well understood. It is not known whether excessive in vivo iron directly contributes to tissue damage or is solely an epiphenomenon. In this article, we focus on the imaging of brain iron and the underlying physiology and metabolism relating to iron deposition. We conclude with a discussion of the potential implications of iron-related toxicity to neurotherapeutic development.

Intranasal administration of a PARG inhibitor profoundly decreases ischemic brain injury.

Cumulative evidence has indicated a critical role of poly(ADP-ribose) polymerase-1 activation in ischemic brain damage. Poly(ADP-ribose) glycohydrolase (PARG) is a key enzyme in poly(ADP-ribose) catabolism. Our previous studies showed that PARG inhibitors, gallotannin (GT) and nobotanin B, can profoundly decrease oxidative cell death in vitro. Here, we tested the hypothesis that intranasal delivery of GT can decrease ischemic brain damage by inhibiting PARG. Intranasal delivery of 25 mg / kg GT within 5 hours after a 2-hour focal brain ischemia markedly decreased the infarct formation and neurological deficits of rats. The GT administration also increased poly(ADP-ribose) in the ischemic brains, suggesting that GT acts as a PARG inhibitor in vivo. Furthermore, the GT treatment abolished nuclear translocation of apoptosis-inducing factor (AIF) in the ischemic brains, suggesting that prevention of AIF translocation may contribute to the protective effects of GT. In contrast, intravenous injection of GT, at 2 hours after ischemic onset, did not reduce ischemic brain damage. Collectively, our findings suggest that PARG inhibition can significantly decrease ischemic brain injury, possibly by blocking AIF translocation. This study also highlights distinct merits of intranasal drug delivery for treating CNS diseases.

Controlled cortical impact in swine: pathophysiology and biomechanics.

Investigations of the basic pathological, cellular, and molecular mechanisms of traumatic brain injury (TBI) over the past two decades have been carried out primarily in rodents. Unfortunately, these studies have not translated into improved outcome in patients with TBI. To better model human TBI, a swine model of controlled cortical impact (CCI) was developed. A CCI device was used to generate a focal lesion in 23 anesthetized male Yorkshire swine. In 10 swine, CCI parameters of velocity and dwell time were varied to achieve a consistent injury (3.5 m/sec, 400 msec, respectively). In 13 swine, depth of depression was varied from 9 to 12 mm. Physiological data, including heart rate (HR), mean arterial blood pressure (MAP), intracranial pressure (ICP), and cerebral perfusion pressure (CPP), were collected for 10 h after injury. Following injury, ICP and HR increased above baseline values in all swine, with a more pronounced elevation in animals impacted to a depth of depression of 12 mm. An 11-mm depth of depression was found to most closely mimic pathological features of human TBI with edema, infiltration of inflammatory cells, pericapillary hemorrhage, and petechial hemorrhages in the white matter. Injury to a depth of depression of 12 mm resulted in cortical laceration obscuring these features. Immunohistological staining with Neu-N, MAP-2, and Fluoro Jade B revealed evidence of degenerating neurons, axonal disruption, and impending cell death. These results indicate that the swine model of CCI results in a defined and reproducible injury with pathological features similar to human TBI. Physiological parameters after injury are readily monitored in a setting mimicking conditions of an intensive care unit, establishing a more clinically relevant experimental model for future investigations.

Human serum albumin and its N-terminal tetrapeptide (DAHK) block oxidant-induced neuronal death.

BACKGROUND AND PURPOSE: Studies using animal models of stroke have shown that human serum albumin (HSA) significantly ameliorates cerebral ischemic injury after both transient and permanent ischemia, even when administered after the onset of ischemia or reperfusion. The mechanism of this effect remains uncertain, and prior studies suggest both indirect hemodynamic and direct cytoprotective effects. HSA is a potent antioxidant, in part because of its strong copper-binding capacity. Here we examined the effect of HSA on oxidant-induced neuronal death in a cortical cell culture system. METHODS: Murine cortical cultures were exposed to oxidative stress generated by hydrogen peroxide and by a mixture of copper plus ascorbic acid. We examined the ability of HSA and a tetrapeptide occupying its N-terminus (DAHK) to prevent neuronal death after these challenges. RESULTS: H(2)O(2) and CuCl(2)/ascorbic acid were used at concentrations that, in the absence of HSA, killed >90% of the neurons. HSA provided complete protection at a concentration of 37.5 micromol/L and 50% protection at 3.75 micromol/L. The copper-binding tetrapeptide DAHK had nearly identical potency and efficacy. HSA and DAHK were also equally effective in preventing neuronal death induced by CuCl(2)/ascorbic acid. CONCLUSIONS: HSA has potent antioxidant properties, probably due to binding of copper and other transition metals. HSA extravasation into ischemic brain may provide neuroprotection by limiting metal-catalyzed oxidant stress. The tetrapeptide DAHK may be an effective, small-molecular-weight alternative to HSA as a therapeutic agent for stroke.

Overexpression of rat heat shock protein 70 reduces neuronal injury after transient focal ischemia, transient global ischemia, or kainic acid-induced seizures.

OBJECTIVE: Transgenic (Tg) mice overexpressing rat heat shock protein 70 (hsp70) demonstrated less infarction than did wild-type (WT) littermates after permanent focal cerebral ischemia. The purpose of this study was to determine whether neuronal injury and apoptosis were reduced in hsp70 Tg mice after transient focal ischemia. The effects of hsp70 overexpression were also evaluated after transient global ischemia or kainic acid (KA)-induced seizures, to verify the results in other excitotoxic stress models. METHODS: Transient focal ischemia was produced with middle cerebral artery occlusion via intraluminal suture cannulation. Infarction volumes were assessed 24 hours after 30 minutes of middle cerebral artery occlusion. Transient global ischemia was produced with 25 minutes of bilateral common carotid artery occlusion. KA (30 mg/kg) was administered subcutaneously, and seizure activity was evaluated. The number of eosinophilic neurons was assessed in the CA1 region 72 hours after bilateral common carotid artery occlusion and in the CA3 region 24 hours after KA administration. RESULTS: The infarction volume after transient middle cerebral artery occlusion was significantly smaller in hsp70 Tg mice than in WT mice (9.1 +/- 5.7 mm(3) versus 22.4 +/- 16.8 mm(3), P < 0.05). The number of eosinophilic neurons in the CA1 area after bilateral common carotid artery occlusion and in CA3 after KA injection was significantly lower in hsp70 Tg mice than in WT mice (949.1 +/- 1095.5 versus 2406.9 +/- 1380.3, P < 0.05, and 33.8 +/- 45.3 versus 119.4 +/- 112.1, P < 0.05, respectively). Fewer terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling-positive cells were observed in hsp70 Tg mice than in WT mice in each model. CONCLUSION: The results demonstrate that overexpression of hsp70 reduces neuronal injury after ischemia and seizures. The reduction in the number of terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling-positive cells in hsp70 Tg mice suggests that hsp70 overexpression might reduce apoptotic cell death.

Overexpression of rat heat shock protein 70 is associated with reduction of early mitochondrial cytochrome C release and subsequent DNA fragmentation after permanent focal ischemia.

Although protective effects of heat shock protein 70 (HSP70) overproduction after ischemic injury have been shown both in vitro and in vivo in neurons, the mechanisms are not fully understood. The hypothesis of this study is that transgenic mice overexpressing HSP70 (HSP70 Tg) show reduced mitochondrial cytochrome c release into cytosol and diminished apoptotic cell death after permanent focal ischemia in comparison to wild-type (Wt) mice. Permanent middle cerebral artery occlusion (pMCAO) was produced by intraluminal suture cannulation in HSP70 Tg and Wt mice. DNA fragmentation was evaluated with DNA gel electrophoresis and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) 24 h after pMCAO. Mitochondrial cytochrome c release into cytosol was assessed with Western blotting and immunohistochemistry 4 h after pMCAO. Cytochrome c levels in the cytosolic fraction were significantly reduced and immunoreactivity of cytochrome c in both cortex and striatum was significantly less in HSP70 Tg mice compared with Wt mice after 4-h pMCAO. DNA laddering, which was clearly observed in Wt mice, was markedly attenuated in HSP70 Tg mice 24 h after pMCAO. The number of TUNEL-positive cells was significantly reduced in HSP70 Tg mice compared with Wt mice. Results are consistent with an association between overexpression of HSP70 and reduction of cytochrome c release with subsequent DNA fragmentation. This may contribute to the HSP70-mediated neuroprotective effect observed after cerebral ischemia.

Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice.

Problems with the intraluminal suture method for induction of focal cerebral ischemia in genetically altered mice include occurrence of subarachnoid hemorrhage (SAH) and variability of infarct volume. We hypothesized that use of 5-0 curved or 6-0 straight suture for carotid cannulation might decrease SAH and that the application of a microvascular clip to the common carotid artery (CCA) might decrease variability of infarct volume. The purpose of this study is to evaluate and explain the results of these technical modifications. Strain related differences in vascular anatomy were evaluated. Male C57BL/6 mice were divided into two groups for permanent and temporary middle cerebral artery occlusion (MCAO). Results of 5-0 curved suture and 6-0 straight suture insertion with and without CCA clip application were examined. Cerebral perfusion was monitored by laser-Doppler flowmetry and infarct volume was measured. After permanent MCAO, larger and more consistent infarct volumes resulted using CCA clip application with a 6-0 but not with a 5-0 suture. After temporary MCAO, the SAH rate was 12.5% with a 5-0 curved suture and 11.1% with a 6-0 straight suture. A 40% rate was observed in a pilot study with 5-0 straight suture. Infarct volume after temporary MCAO with a CCA clip was significantly larger and variability of infarct volume was smaller than without the CCA clip using 5-0 curved and 6-0 straight suture. In summary, SAH is less frequent using a 5-0 curved or 6-0 straight suture. Infarct volume is enlarged by application of a CCA clip (249).

Mild hypothermia reduces zinc translocation, neuronal cell death, and mortality after transient global ischemia in mice.

The authors sought to determine whether Zn translocation associated with neuronal cell death occurs after transient global ischemia (TGI) in mice, as has been previously shown in rats, and to determine the effect of mild hypothermia on this reaction. To validate the TGI model, carbon-black injection and laser-Doppler flowmetry were compared in three strains of mice (C57BL/6, SV129, and HSP70 transgenic mice) to assess posterior communicating artery (PcomA) development and cortical perfusion. In C57BL/6 mice, optimal results were obtained when subjected to 20-minute TGI. Brain and rectal temperature measurements were compared to monitor hypothermia. Results of TGI were compared in normothermia (NT; 37 degrees C) and mild hypothermia groups (HT; 33 degrees C) by staining with Zn -specific fluorescent dye, -(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) and hematoxylin-eosin 72 hours after reperfusion. The Zn translocation observed in hippocampus CA1, CA2, and Hilus 72 hours after 20 minutes of TGI was significantly reduced by mild hypothermia. The number of degenerating neurons in the HT group was significantly less than in the NT group. Mild hypothermia reduced mortality significantly (7.1% in HT, 42.9% in NT). Results suggest that mild hypothermia may reduce presynaptic Zn release in mice, which protects vulnerable hippocampal neurons from ischemic necrosis. Future studies may further elucidate mechanisms of Zn -induced ischemic injury.