Severity profile of penetrating ballistic-like brain injury on neurofunctional outcome, blood-brain barrier permeability, and brain edema formation

This study evaluated the injury severity profile of unilateral, frontal penetrating ballistic-like brain injury (PBBI) on neurofunctional outcome, blood-brain barrier (BBB) permeability, and brain edema formation. The degree of injury severity was determined by the delivery of a water-pressure pulse designed to produce a temporary cavity by rapid (<40?ms) expansion of the probe's elastic balloon calibrated to equal 5%, 10%, 12.5%, or 15% of total rat brain volume (control groups consisted of sham surgery or insertion of the probe only). Neurofunctional assessments revealed motor and cognitive deficits related to the degree of injury severity, with the most clear-cut profile of PBBI injury severity depicted by the Morris water maze (MWM) results. A biphasic pattern of BBB leakage was detected in the injured hemisphere at all injury severity levels at 4?h post-injury, and again at 48-72?h post-injury, which remained evident out to 7 days post-PBBI in the 10% and 12.5% PBBI groups. Likewise, significant brain edema was detected in the injured hemisphere by 4?h post-injury and remained elevated out to 7 days post-injury in the 10% and 12.5% PBBI groups. However, following 5% PBBI, significant levels of edema were only detected from 24?h to 48h post-injury. These results identify an injury severity profile of BBB permeability, brain edema, and neurofunctional impairment that provides sensitive and clinically relevant outcome metrics for studying potential therapeutics.     

血红素加氧酶-1对心肺复苏诱发大鼠脑水肿发生的影响

目的 探讨血红素加氧酶-1(HO-1)对心肺复苏诱发大鼠脑水肿发生的影响.方法 雄性SD大鼠40只,随机分为4组(n=10):假手术组(S组)、心肺复苏组(CAR组)、氯化高铁血红素组(H组)和锡原卟啉组(SnPP组).CAR组、H组和SnPP组进行窒息性心跳骤停后行心肺复苏,H组预先12 h经腹腔注射氯化高铁血红素15 mg/kg,SnPP组预先1 h经腹腔注射锡原卟啉Ⅸ30 μmol/kg.各组于恢复自主循环(ROSC)后1 h和6 h时断头取脑组织,测定脑皮质、海马和脑干含水量,测定HO-1和水通道蛋白4(AQP4)mRNA表达.结果 与S组比较,CAR组和SnPP组皮质和海马含水量增加,海马AQP4 mRNA表达上调,H组HO-1 mRNA表达上调,SnPP组HO-4 mRNA表达下调(P<0.05);与CAR组比较,H组皮质和海马含水量降低,皮质及海马AQP4 mRNA表达下调,HO-1 mRNA表达上调(P<0.05),SnPP组T_2时皮质及海马AQP4 mRNA表达上调,HO-1 mRNA表达下调(P<0.05),各组脑干含水量比较差异无统计学意义(P>0.05).结论 HO-1活性增高可减轻大鼠心肺复苏早期脑水肿,其机制可能与下调AQP4的表达有关.     

CYP4Fs expression in rat brain correlates with changes in LTB4 levels after traumatic brain injury

Cytochrome P450 (CYP) 4Fs constitute a subgroup of the cytochrome P450 superfamily and are involved in cellular protection and metabolism of numerous molecules, including drugs, toxins, and eicosanoids. CYP4Fs are widely distributed in rat brain with each isoform having a unique distribution pattern throughout different brain regions. The present study shows that traumatic brain injury (TBI) triggers inflammation and elicits changes in mRNA expression of CYP4Fs in the frontal and occipital lobes and the hippocampus. At 24 h post-injury, almost all CYP4F mRNA expression is suppressed compared with sham control throughout these three regions, while at 2 weeks post-injury, all CYP4F mRNAs increase, reaching levels higher than those at 24 h post-injury or uninjured controls. These changes in CYP4F levels inversely correlate with levels of leukotriene B4 (LTB4) levels in the brain following injury at the same time points. TBI also causes changes in CYP4F protein expression and localization around the injury site. CYP4F1 and CYP4F6 immunoreactivity increases in surrounding astrocytes, while CYP4F4 immunoreactivity shifts from endothelia of cerebral vessels to astrocytes.     

Brain edema after experimental intracerebral hemorrhage: role of hemoglobin degradation products

OBJECT: The mechanisms involved in brain edema formation following intracerebral hemorrhage (ICH) have not been fully elucidated. The authors have found that red blood cell lysis plays an important role in edema development after ICH. In the present study, they sought to determine whether degradation products of hemoglobin cause brain edema. METHODS: Hemoglobin, hemin, bilirubin, or FeCl2 were infused with stereotactic guidance into the right basal ganglia of Sprague-Dawley rats. The animals were killed 24 hours later to determine brain water and ion contents. Western blot analysis and immunohistochemistry were applied for heme oxygenase-1 (HO-1) measurement. The effects of an HO inhibitor, tin-protoporphyrin (SnPP), and the iron chelator deferoxamine, on hemoglobin-induced brain edema were also examined. Intracerebral infusion of hemoglobin, hemin, bilirubin, or FeCl2 caused an increase in brain water content at 24 hours. The HO-1 was upregulated after hemoglobin infusion and HO inhibition by SnPP-attenuated hemoglobin-induced edema. Brain edema induced by hemoglobin was also attenuated by the intraperitoneal injection of 500 mg/kg deferoxamine. CONCLUSIONS: Hemoglobin causes brain edema, at least in part, through its degradation products. Limiting hemoglobin degradation coupled with the use of iron chelators may be a novel therapeutic approach to limit brain edema after ICH.     

Overexpression of SSAT in kidney cells recapitulates various phenotypic aspects of kidney ischemia-reperfusion injury

To ascertain the role of spermidine/spermine N-1-acetyl-transferase (SSAT; the rate-limiting enzyme in polyamine catabolism) in cell injury, cultured kidney (HEK 293) cells conditionally overexpressing SSAT were generated. The SSAT expression was induced and its enzymatic activity increased 24 h after addition of tetracycline and remained elevated over the length of the experiments. Induction of SSAT upregulated the expression of polyamine oxidase and resulted in the reduction of cellular concentration of spermidine and spermine, increased concentration of putrescine, and inhibited cell growth. SSAT overexpression increased the expression of heme oxygenase-1 (HO-1) by 350% 24 h after addition of tetracycline, indicating the induction of oxidative stress. The presence of catalase significantly prevented the upregulation of HO-1 in SSAT overexpressing cells, indicating that generation of H2O2 is partially responsible for the induction of oxidative stress. Overexpression of SSAT caused rounding and loss of cell anchorage and significantly altered the morphology of actin-containing filopodia, suggesting an adhesion defect. SSAT upregulation may mediate majority of the oxidative stress in kidney ischemia-reperfusion injury (IRI) as manifested by decreased cell growth, generation of toxic metabolites (H2O2 and putrescine), upregulation of HO-1, disruption of cell anchorage, and defect in cell adhesion. These data point to the inhibition of polyamine catabolism as a therapeutic approach for the prevention of tissue injury in kidney IRI.     

血红素氧合酶-1在临床肝移植中肝脏保护作用的研究

目的 研究移植肝脏血红素氧合酶(HO-1)表达水平与缺血再灌注损伤和移植术后肝脏功能的关系.方法 研究28例人类临床原位肝脏移植,根据供肝血红素氧合酶(HO-1)表达的平均值将供肝分为两组:移植前供肝HO-1高表达组和移植前供肝HO-1低表达组.比较两组移植术后血浆AST、ALT水平、胆汁中胆盐含量以及术前术后HO-1 mRNA和蛋白表达情况.结果 再灌注后移植术前HO-1低表达组的HO-1 mRNA表达显著增加,而高表达组HO-1 mRNA表达却有所下降.肝脏移植后,术前HO-1低表达组与高表达组相比,血浆转氨酶显著降低,胆汁中胆盐含量明显高于后者.结论 移植术前HO-1低表达组供肝在再灌注过程中能够进一步诱导HO-1表达,与高表达组供肝相比其所遭受的缺血再灌注损伤较轻,移植术后肝脏功能较好.移植过程中HO-1表达的增强要比移植前HO-1高表达更具有细胞保护作用.免疫荧光染色证实枯否细胞是人类肝脏表达HO-1的主要部位.     

Cyclin D1 gene ablation confers neuroprotection in traumatic brain injury

Cell cycle activation (CCA) is one of the principal secondary injury mechanisms following brain trauma, and it leads to neuronal cell death, microglial activation, and neurological dysfunction. Cyclin D1 (CD1) is a key modulator of CCA and is upregulated in neurons and microglia following traumatic brain injury (TBI). In this study we subjected CD1-wild-type (CD1(+/+)) and knockout (CD1(-/-)) mice to controlled cortical impact (CCI) injury to evaluate the role of CD1 in post-traumatic neurodegeneration and neuroinflammation. As early as 24?h post-injury, CD1(+/+) mice showed markers of CCA in the injured hemisphere, including increased CD1, E2F1, and proliferating cell nuclear antigen (PCNA), as well as increased Fluoro-Jade B staining, indicating neuronal degeneration. Progressive neuronal loss in the hippocampus was observed through 21 days post-injury in these mice, which correlated with a decline in cognitive function. Microglial activation in the injured hemisphere peaked at 7 days post-injury, with sustained increases at 21 days. In contrast, CD1(-/-) mice showed reduced CCA and neurodegeneration at 24?h, as well as improved cognitive function, attenuated hippocampal neuronal cell loss, decreased lesion volume, and cortical microglial activation at 21 days post-injury. These findings indicate that CD1-dependent CCA plays a significant role in the neuroinflammation, progressive neurodegeneration, and related neurological dysfunction resulting from TBI. Our results further substantiate the proposed role of CCA in post-traumatic secondary injury, and suggest that inhibition of CD1 may be a key therapeutic target for TBI.     

Acute subdural hematoma associated with diffuse brain injury and hypoxemia in the rat: effect of surgical evacuation of the hematoma

The aim of this study was to assess the effect of rapid or delayed surgical evacuation on the physiological consequence and brain edema formation in a rat model of acute subdural hematoma (SDH) coupled with either diffuse brain injury (DBI) or hypoxemia. The SDH was made by an autologous blood injection, while DBI was induced using the impact acceleration model (mild, 450 g/1 m; severe, 450 g/2 m). Physiological parameters measured included intracranial pressure (ICP), mean arterial blood pressure (MABP), cerebral blood flow (CBF), and brain tissue water content. At 1 h (rapid evacuation) or 4 h (delayed evacuation) after the SDH induction, surgical evacuation following a craniotomy was performed using saline irrigation and forceps. The study consisted of three different series, including 400 microL of SDH alone (Series 1), SDH400 + mild DBI (Series 2), and SDH300 + severe DBI + 20 min hypoxemia (Series 3). The hypoxemia was added in Group 3 to produce a steadily increasing ICP. In Series 1 and 2, all rats were randomized into the three following groups: non-, rapid, and delayed evacuation; Series 3 had two groups: non- and rapid evacuation. In Series 1, the surgical evacuation showed no beneficial effects on the brain edema formation assessed at 5 h post-injury. In Series 2, the rapid, but not delayed, evacuation significantly reduced both the increased ICP level and brain water content. The additional insult of hypoxemia (Series 3) resulted in a progressive ICP elevation, persistently depressed CBF, and severe brain swelling. Under this situation, the rapid evacuation exacerbated brain edema. These results have clinical implications for the management of severe traumatic SDH, especially its operative indication and timing.     

Treatment window for hypothermia in brain injury.

OBJECT: The goal of this study was to evaluate the therapeutic window for hypothermia treatment following experimental brain injury by measuring edema formation and functional outcome. METHODS: Traumatic brain injury (TBI) was produced in anesthetized rats by using cortical impact injury. Edema was measured in the ipsilateral and contralateral hemispheres by subtracting dry weight from wet weight, and neurological function was assessed using a battery of behavioral tests 24 hours after TBI. In injured rats, it was found that brain water levels were elevated at I hour postinjury, compared with those in sham-injured control animals, and that edema peaked at 24 hours and remained elevated for 4 days. Hypothermia (3 hours at 30 degrees C) induced either immediately after TBI or 60 minutes after TBI significantly reduced early neurological deficits. Delay of treatment by 90 or 120 minutes postinjury did not result in this neurological protection. Immediate administration of hypothermia also significantly decreased the peak magnitude of edema at 24 hours and 48 hours postinjury, compared with that in normothermic injured control animals. When delayed by 90 minutes, hypothermia did not affect the pattern of edema formation. CONCLUSIONS: When hypothermia was administered immediately or 60 minutes after TBI, injured rats showed an improvement in functional outcome and a decrease in edema. Delayed hypothermia treatment had no effect on functional outcome or on edema.     

Penetrating ballistic-like brain injury in the rat: differential time courses of hemorrhage, cell death, inflammation, and remote degeneration

Acute and delayed cerebral injury was assessed in a recently developed rat model of a penetrating ballistic-like brain injury (PBBI). A unilateral right frontal PBBI trajectory was used to induce survivable injuries to the frontal cortex and striatum. Three distinct phases of injury progression were observed. Phase I (primary injury, 0-6 h) began with immediate (<5 min) intracerebral hemorrhage (ICH) that reached maximal volumetric size at 6 h (27.0 +/- 2.9 mm(3)). During Phase II (secondary injury, 6-72 h), a core lesion of degenerate neurons surrounding the injury track expanded into peri-lesional areas to reach a maximal volume of 69.9 +/- 6.1 mm(3) at 24 h. The core lesion consisted of predominately necrotic cell death and included marked infiltration of both neutrophils (24 h) and macrophages (72 h). Phase III (delayed degeneration, 3-7 days) involved the degeneration of neurons and fiber tracts remote from the core lesion including the thalamus, internal capsule, external capsule, and cerebral peduncle. Overall, different time courses of hemorrhage, lesion evolution, and inflammation were consistent with complementary roles in injury development and repair, providing key information about these mediators of primary, secondary, and delayed brain injury development. The similarities/differences of PBBI to other focal brain injury models are discussed.     

Inhibition of ischemia/reperfusion injury and chronic graft deterioration by a single-donor treatment with cobalt-protoporphyrin for the induction of heme oxygenase-1

Today, the major problem in organ transplantation is not acute graft rejection but chronic graft deterioration. In addition to alloantigen-specific events, alloantigen independent factors like donor age, previous diseases, consequences of brain death, and perioperative events of ischemia/reperfusion injury have a major impact on long-term graft function. The induction of the stress protein heme oxygenase-1 (HO-1) protects cells from injury and apoptosis. Here, we tested the protective effects of HO-1 induction in a clinically relevant kidney transplant model. Induction of HO-1 expression following cobalt-protoporphyrin (CoPP) treatment in organ donors prolonged graft survival and long-term function remarkably following extended periods of ischemia. Positive effects were observed with both optimal and marginal grafts from old donor animals. Structural changes characteristic for chronic rejection, as well as graft infiltration by monocytes/macrophages and CD8+ T cells, were substantially reduced following HO-1 induction. Up-regulation of HO-1 expression before organ transplantation was also associated with reduced levels for tumor necrosis factor (TNF)-alpha mRNA, increased levels for interferon (IFN)-gamma, and bcl-x, and insignificant differences for CD25, interleukin (IL)-2, IL-4, IL-6, and IL-10 mRNA levels. The significant improvement of long-term graft function following induction of HO-1 expression in donor organs suggests that this strategy may be a novel clinical treatment option with particular relevance for transplantation of marginal organs.     

Extraluminal cooling of bilateral common carotid arteries as a method to achieve selective brain cooling for neuroprotection

Systemic cooling to achieve brain hypothermia has been investigated as a neuroprotective therapy but can present serious adverse effects. Here we describe a novel method to selectively cool the rat brain and investigate its neuroprotective effects following transient middle cerebral artery occlusion (MCAo). The novelty of our method of selective brain cooling (SBC) was that the extraluminal cooling of the carotid arterial blood was achieved by using a cooling cuff wrapped around each common carotid artery (CCA). Within 20 min of CCA cooling, brain temperature could be lowered by 2-5 degrees C below the baseline and maintained stable for approximately 2 h while maintaining body temperature at 37 degrees C. No adverse effects of SBC were observed on systemic physiology, regional cerebral blood flow (rCBF), bleeding time, or tissue histology in normal animals. In rats having sustained 2-h MCAo, intra-ischemic SBC for 90 min, initiated 30 min following the onset of ischemia, significantly reduced infarction measured at 24 h post-injury (normothermic rats=312+/-51 mm3, SBC rats=139+/-83 mm3). In subgroup experiments, the incidence of peri-infarct depolarization (PID) was assessed during the MCAo and cooling period. Compared to normothermic but ischemic rats, SBC significantly reduced the number of PID events from 6.2+/-2.5 to 2.0+/-2.5, and reduced infarct volumes from 323+/-79 to 139+/-102 mm3. In conclusion, this extralumimal cooling method of SBC provides a safe and efficient approach to rapidly and safely achieve hypothermic neuroprotection.     

Concussive brain trauma in the mouse results in acute cognitive deficits and sustained impairment of axonal function

Concussive brain injury (CBI) accounts for approximately 75% of all brain-injured people in the United States each year and is particularly prevalent in contact sports. Concussion is the mildest form of diffuse traumatic brain injury (TBI) and results in transient cognitive dysfunction, the neuropathologic basis for which is traumatic axonal injury (TAI). To evaluate the structural and functional changes associated with concussion-induced cognitive deficits, adult mice were subjected to an impact on the intact skull over the midline suture that resulted in a brief apneic period and loss of the righting reflex. Closed head injury also resulted in an increase in the wet weight:dry weight ratio in the cortex suggestive of edema in the first 24 h, and the appearance of Fluoro-Jade-B-labeled degenerating neurons in the cortex and dentate gyrus of the hippocampus within the first 3 days post-injury. Compared to sham-injured mice, brain-injured mice exhibited significant deficits in spatial acquisition and working memory as measured using the Morris water maze over the first 3 days (p<0.001), but not after the fourth day post-injury. At 1 and 3 days post-injury, intra-axonal accumulation of amyloid precursor protein in the corpus callosum and cingulum was accompanied by neurofilament dephosphorylation, impaired transport of Fluoro-Gold and synaptophysin, and deficits in axonal conductance. Importantly, deficits in retrograde transport and in action potential of myelinated axons continued to be observed until 14 days post-injury, at which time axonal degeneration was apparent. These data suggest that despite recovery from acute cognitive deficits, concussive brain trauma leads to axonal degeneration and a sustained perturbation of axonal function.     

Neurological recovery-promoting, anti-inflammatory, and anti-oxidative effects afforded by fenofibrate, a PPAR alpha agonist, in traumatic brain injury

We previously demonstrated that fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, reduced the neurological deficit, the edema and the cerebral lesion induced by traumatic brain injury (TBI). In order to elucidate these beneficial effects, in the present study, we investigated, in the same TBI model, fenofibrate's effects on the inflammation and oxidative stress. Male Sprague Dawley rats were randomized in four groups: non-operated, sham-operated, TBI + vehicle, TBI + fenofibrate. TBI was induced by lateral fluid percussion of the temporoparietal cortex. Rats were given fenofibrate (50 mg/kg) or its vehicle (water containing 0.2% methylcellulose), p.o. 1 and 6 h after brain injury. A neurological assessment was done 24 h after TBI, then rats were killed and the brain COX2, MMP9 expression, GSx, GSSG levels were determined. The same schedule of treatment was used to evaluate the effect of fenofibrate on immunohistochemistry of 3NT, 4HNE and iNOS at 24 h post-injury. Our results showed that fenofibrate promotes neurological recovery by exerting anti-inflammatory effect evidenced by a decrease in iNOS, COX2 and MMP9 expression. In addition, fenofibrate showed anti-oxidant effect demonstrated by a reduction of markers of oxidative stress: loss of glutathione, glutathione oxidation ratio, 3NT and 4HNE staining. Our data suggest that PPARalpha activation could mediate pleiotropic effects and strengthen that it could be a promising therapeutic strategy for TBI.     

Plasma and urinary heme oxygenase-1 in AKI

AKI induces upregulation of heme oxygenase 1 (HO-1), which exerts cytoprotective effects and modulates the renal response to injury, suggesting that a biomarker of intrarenal HO-1 activity may be useful. Because HO-1 largely localizes to the endoplasmic reticulum and has no known secretory pathway, it is unclear whether plasma or urinary levels of HO-1 reflect intrarenal HO-1 expression. We measured plasma and urinary levels of HO-1 by ELISA during the induction and/or maintenance phases of four mouse models of AKI: ischemia/reperfusion, glycerol-induced rhabdomyolysis, cisplatin nephrotoxicity, and bilateral ureteral obstruction. In addition, we measured levels of HO-1 mRNA and protein in the renal cortex. Each AKI model increased renal HO-1 gene expression, which corresponded with release of HO-1 into plasma and urine by 4 hours. Over time, the magnitudes of plasma and urinary HO-1 paralleled renal cortical gene expression. AKI and the associated uremia did not seem to affect extrarenal HO-1 gene activity assessed in the liver, lung, and spleen. In iron-challenged, cultured proximal tubule cells, we observed a positive correlation between HO-1 mRNA level and HO-1 release. In humans, 10 patients with AKI demonstrated markedly higher levels of plasma and urine HO-1 levels than 10 critically ill patients without AKI or 20 patients with CKD or ESRD. In summary, these data suggest that plasma and urinary HO-1 levels may serve as biomarkers of AKI and intrarenal HO-1 gene activity.