Original Article

Journal of Cerebral Blood Flow & Metabolism (2000) 20, 531–542; doi:10.1097/00004647-200003000-00012

Importance of Posttraumatic Hypothermia and Hyperthermia on the Inflammatory Response After Fluid Percussion Brain Injury: Biochemical and Immunocytochemical Studies

This study was supported by National Institutes of Health grant number NS-30291.

Katina Chatzipanteli*, Ofelia F Alonso*, Susan Kraydieh† and W Dalton Dietrich*,†

  1. *Departments of Neurology, The Neurotrauma Research Center Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida, U.S.A.
  2. †Neurological Surgery, The Neurotrauma Research Center Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida, U.S.A.

Correspondence: W. Dalton Dietrich, The Miami Project to Cure Paralysis (R48), University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, U.S.A.

Received 23 September 1999; Revised 25 November 1999; Accepted 26 November 1999.

Top

Abstract

The purpose of this study was to investigate: 1) the temporal and regional profile of polymorphonuclear leukocyte (PMNL) infiltration after moderate traumatic brain injury using the parasagittal fluid percussion model and 2) the effects of posttraumatic hypothermia (30°C) and hyperthermia (39°C) on the acute and subacute inflammatory response. We hypothesized that posttraumatic hypothermia would reduce the degree of PMNL accumulation whereas hyperthermia would exacerbate this response to injury. In the first series of experiments we quantitated the temporal profile of altered myeloperoxidase activity under normothermic (37°C) conditions (n = 20). The rats were allowed to survive for 3 hours, 24 hours, 3 days, or 7 days after trauma, and brains were dissected into cortical and subcortical regions ipsilateral and contralateral to injury. Additional animals were perfused and fixed for the immunocytochemical visualization of myeloperoxidase (n = 15). In the second series of experiments, rats (n = 25) were killed 3 hours or 3 days after the 3-hour monitoring period of normothermia (36.5°C), hypothermia (30°C), or hyperthemia (39°C) (n = 4 to 5 per group), and myeloperoxidase activity was again quantitated. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased (P < 0.05) at 3 hours within the anterior cortical segment (213.97 plusminus 56.2 versus control 65.5 plusminus 52.3 U/g of wet tissue; mean plusminus SD) and posterior (injured) cortical and subcortical segments compared to sham-operated rats (305.76 plusminus 27.8 and 258.67 plusminus 101.4 U/g of wet tissue versus control 62.8 plusminus 24.8 and 37.28 plusminus 35.6 U/g of wet tissue; P < 0.0001, P < 0.05, respectively). At 24 hours and 7-days after trauma only the posterior cortical region (P < 0.005, P < 0.05, respectively) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was also significantly increased within the anterior cortical segment (P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex (P < 0.0001, P < 0.05, respectively). Immunocytochemical analysis of myeloperoxidase reactivity at 3 hours, 24 hours, 3- and 7-days demonstrated large numbers of immunoreactive leukocytes within and associated with blood vessels, damaged tissues, and subarachnoid spaces. Posttraumatic hypothermia and hyperthermia had significant effects on myeloperoxidase activity at both 3 hours and 3 days after traumatic brain injury. Posttraumatic hypothermia reduced myeloperoxidase activity in the injured and noninjured cortical and subcortical segments compared to normothermic values (P < 0.05). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior cortical region compared to normothermic values at both 3 hours and 3 days (473.5 plusminus 258.4 and 100.11 plusminus 27.58 U/g of wet tissue, respectively, P < 0.05 versus controls). These results indicate that posttraumatic hypothermia decreases early and more prolonged myeloperoxidase activation whereas hyperthermia increases myeloperoxidase activity. Temperature-dependent alterations in PMNL accumulation appear to be a potential mechanism by which posttraumatic temperature manipulations may influence traumatic outcome.

Keywords:

Myeloperoxidase, Fluid percussion injury, Rat, Temperature

Abbreviations:

BBB, blood-brain barrier; CCI, controlled cortical impact; F-P, fluid percussion; PMNL, polymorphonuclear leukocyte; TBI, traumatic brain injury

Ischemic and traumatic brain injury (TBI) lead to inflammatory events that are believed to contribute to outcome through secondary injury mechanisms (Hallenbeck et al., 1986; Giulian et al., 1989; Shoettlle et al. 1990; Kochanek et al., 1992; Biagas et al., 1992; Chopp et al., 1994; Clark et al., 1994; del Zoppo and Garcia, 1995; Tomina and Fukuuchi, 1996; Hartl et al., 1997; Jean et al., 1998; Grady et al., 1999). Among the known critical factors that are involved in the spread of secondary damage after TBI is the acute inflammatory response to injury involving the infiltration and accumulation of polymorphonuclear leukocytes (PMNLs). Potential consequences of PMNL activation and accumulation include increased microvascular permeability (Petito, 1979; Wahl et al., 1988; Cortez et al., 1989; Beynon et al., 1993), edema formation (Schoettle et al., 1990; Bareyre et al., 1997), intracranial hypertension (Uhl et al., 1994), and secondary neuronal injury (Chopp et al., 1994).

In various models of cerebral ischemia (Barone et al., 1991; Chopp et al., 1994), the involvement of PMNL recruitment as contributors to the acute inflammatory response has been reported (for reviews see Kochanek and Hallenbeck, 1992; Jean et al., 1998). Neutrophils have been reported to be the first inflammatory cells to arrive at the ischemic injury site, followed by the infiltration of mononuclear phagocytes (Schoettle et al., 1990; Clark et al., 1994). In models of TBI including weight drop, control cortical impact, intracerebral stab injury, and fluid percussion (F-P), an influx of neutrophil accumulation has been shown as early as 4 to 8 hours after trauma with peak values occurring at 48 hours (Persson 1976; Biagas et al., 1992; Horner et al., 1992; Clark et al., 1994; Dietrich et al., 1996b; Toyoda et al., 1996; Carlos et al., 1997). The infiltration and accumulation of activated PMNLs into extravascular tissues depend on PMNLs-endothelial interactions between cell surface leukocyte integrins and adhesion molecules such as endothelial selectins (E- and P- selectin). Increased expression of intercellular adhesion molecule (ICAM-1) and platelet endothelial adhesion molecule-1 (PECAM-1) after brain trauma injury account partly for neutrophil adhesion to endothelial cells and transendothelial migration (Zhang et al., 1994b; Carlos et al., 1997; Whalen et al., 1997a, 1997b, 1999).

After injury, activated neutrophils, macrophages, and disrupted endothelial cells produce a variety of cytokines that may also contribute to the pathologic process (Zimmerman, 1986; Lindholm et al., 1987; Hama et al., 1989; David et al., 1990; Marion et al., 1993; Taupin et al., 1993; Ott et al., 1994; Fan et al., 1995, 1996; Goss et al., 1995; Shohami et al., 1996, 1997; Mantovani et al., 1997). Cytokines are capable of altering vascular permeability (Beynon et al., 1993) and exacerbate subsequent brain edema formation (Albelda et al., 1994; Matsuo et al., 1994a, 1994b). These events may lead to the influx of more inflammatory cells, with the resultant production of harmful reactive oxygen species (Hallenbeck et al., 1986; Kochanek et al., 1987; Saito et al., 1988; Ward et al., 1988; Grogaard et al., 1989; Barone et al., 1991; Clark et al., 1994; Matsuo 1995).

Posttraumatic hypothermia has been shown in several models of TBI to improve histopathologic and behavioral outcome (Jiang et al., 1991; Arnould et al., 1993; Clifton et al., 1993; Lyeth et al., 1993; Palmer et al., 1993; Dietrich et al., 1994a, 1994b, 1996c; Bramlett et al., 1995; Globus et al., 1995; Clark et al., 1996; Marion et al., 1997). In brain trauma models, posttraumatic hypothermia has been reported to preserve the integrity of the blood-brain barrier (BBB) (Jiang et al., 1992), reduce neuronal injury, decrease contusion volume (Dietrich et al., 1990b; Palmer et al., 1993), and reduce cerebral oxygen consumption (Rosomoff and Haladay, 1954). Cytoskeletal components including microtubule-associated protein have been reported to be protected from TBI by hypothermia (Taft et al., 1993). In addition, hypothermia applied after TBI attenuates the increase in extracellular levels of excitatory amino acids and oxygen-free radical production (Globus et al., 1995). Recent reports using the control cortical impact model of TBI demonstrated that PMNL accumulation in the injured cortex was significantly decreased in rats maintained at 32°C versus 39°C at 4 hours after TBI (Whalen et al., 1997a, 1997b).

In contrast to hypothermia, hyperthermia has been shown to aggravate outcome in models of brain injury (Dietrich et al., 1996b). After cerebral ischemia for example, hyperthermia has been reported to worsen neuronal and cerebrovascular outcome (Dietrich et al., 1990a, 1990b; Chen et al., 1991; Morikawa et al., 1992). In TBI, hyperthermia (39°C) applied 24 hours after F-P injury increased contusion volume, BBB breakdown, axonal injury, and mortality (Dietrich et al., 1996b). Despite extensive data indicating the beneficial effects of posttraumatic hypothermia on outcome and the detrimental effects of posttraumatic hyperthermia, the effect of posttraumatic temperature manipulations on the acute and subacute inflammatory response to F-P brain injury has not been investigated.

To quantitate the accumulation of PMNLs at the injury site, we conducted a regional determination of myeloperoxidase activity which has been reported to be a reliable marker of PMNL accumulation (Barone et al., 1991; Zhang and Chopp, 1997). Immunocytochemical studies were conducted in parallel to document the regional distribution of neutrophils in the traumatized brain. Our findings demonstrate that PMNLs accumulate within 3 hours after moderate F-P injury within the histopathologically vulnerable brain regions. In addition posttraumatic temperature modifications significantly influence the inflammatory response to TBI.

Top

MATERIALS AND METHODS

Animal groups

The myeloperoxidase enzymatic activity was performed on 20 male Sprague-Dawley rats weighing between 300 and 400 g obtained from Charles River Breeders. They were kept at a constant temperature (approx25°C) in an air-conditioned room for at least 7 days before the study and exposed to a 12-hour light-dark cycle. Rats were allowed free access to food and water before and after the surgery. The studies were conducted in two phases. In the first phase, the temporal profile of myeloperoxidase activity was determined: group 1, the normothermic group, time = 3 hours, 24 hours, 3 days, or 7 days and sham (n = 3 in each group). The same temporal profile of immunocytochemical detection of myeloperoxidase was performed on 12 male Sprague-Dawley rats weighing between 300 and 400 g (n = 4 in each group). Sham-operated control rats underwent all surgical procedures but were not traumatized and killed 24 hours later. In the second phase, the effects of posttraumatic hypothermia or hyperthermia on myeloperoxidase activity at 3 hours and 3 days were assessed: group 2, normothermic (37°C) group 3-hour survival (n = 4); group 3, hypothermic (30°C) group 3-hour survival (n = 4); group 4, hyperthermic (39°C) group 3-hour survival (n = 5); group 5, 3 hours of posttraumatic normothermia and 3-days survival (n = 4); group 6, 3 hours of posttraumatic hypothermia and 3 days survival (n = 4); group 7, 3-hour posttraumatic hyperthermic group and 3-days survival (n = 4). After the 3-hour temperature monitoring period the animals were allowed to recover and had free access to food and water. The 3-hour period was chosen because this represented the earliest time after the temperature monitoring period that could be assessed. The 3-day period represented the maximal myeloperoxidase response after TBI.

Surgical preparation

All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. The basic surgical preparation for the F-P brain injury was performed according to methods previously described (Dietrich et al., 1994a). Briefly, 1 day before injury, the rats were anesthetized with Equitensin (a mixture of nembutal, propylene glycol, ethanol, MgSO4, and chloral hydrate). Rats were then placed in a stereotaxic frame, and a 4.8 mm craniectomy was made overlying the right parietal cortex (3.8 mm posterior to bregma and 2.5 mm lateral to the midline) (Zilles, 1985). A plastic injury tube was then placed over the exposed dura, was bound by adhesive, and then dental acrylic was poured around the injury tube. After the acrylic had hardened, the scalp was closed, and the animal was returned to its cage and allowed to recover overnight before TBI.

The next day, a fluid percussion (F-P) device was used to produce experimental TBI (Dixon et al., 1987; McIntosh et al., 1989). The rats were reanesthetized with 3.0% halothane in a gas mixture of 70% nitrous oxide and 30% oxygen and maintained at 0.5% halothane. An endotracheal tube was inserted orally, and rats were mechanically ventilated. During ventilation, the animals were paralyzed with pancronium bromide (1 mg/kg per hour, intravenously). Moderate head injury ranging from 1.8 to 2.2 atm was next produced. The F-P device consists of a saline-filled Plexiglass cylinder that is fitted with a transducer housing and injury screw adapted for the rat's skull. The metal screw is firmly connected to the plastic injury tube of the anesthetized rat, and the injury is induced by the descent of a pendulum that strikes the piston. In the first phase of experiments where the temporal profile of myeloperoxidase was assessed, temporalis muscle temperature was monitored as an indirect measure of brain temperature throughout the experiment and maintained at a normothermic (37.0°C) level using a feedback heating lamp above the animal's head (Jiang et al., 1991). Rectal temperature was also monitored and maintained at normothermic levels throughout the study using a feedback heating lamp above the animal's body. Arterial blood pressure was monitored by means of the right femoral artery and recorded for up to 60 minutes after TBI. Blood gases were obtained from arterial samples taken from the right femoral artery just before TBI and 60 minutes later. Blood glucose levels were also monitored. The animals in the sham groups for myeloperoxidase activity measurements were subjected to all of the same surgical procedures except for the actual insult, including anesthesia and the placement of a plastic injury tube.

In the second phase of experiments the effects of posttraumatic hypothermia and hyperthermia on myeloperoxidase activity were assessed. Posttraumatic rats were brought to the target temporalis muscle temperature within 5 minutes and maintained under normothermic (36.62 plusminus 0.086°C), hypothermic (30.18 plusminus 0.14°C), or hyperthermic (39.68 plusminus 0.217°C) conditions for 3 hours after TBI. In animals in which brain temperature was lowered, cold air was blown directly onto the head.

Myeloperoxidase assay

Myeloperoxidase, a heme lysosomal enzyme present in azurophilic granules of neutrophils and in smaller quantities in monocytes, has been commonly used as a marker of PMN accumulation (Barone et al., 1991). The assay method used in this study was a modification of a previously described method by Barone et al., (1991). To eliminate intravascular blood, rats were intracardially perfused with 250 mL of ice-cold 0.9% NaCl (at a pressure of 100 mm Hg). A 3-mm thick coronal brain slice was first made at the bregma levels 1.2 mm and -1.8 mm and included cortical and subcortical areas anterior to the histopathologically vulnerable posterior cortical and subcortical regions (i.e., -1.8 mm and -4.8 mm) using Rodent Brain Matrices (Harvard Apparatus, Inc. MA, U.S.A.) (Fig. 1). The anterior cortical and subcortical regions adjacent to the injury were labeled as ACips and ASCips, respectively. The posterior cortical and subcortical sites that included histopathologically vulnerable regions (i.e., posterior cortical and subcortical segments) were dissected and labeled as PCips and PSCips. The homotopic cortical and subcortical regions of the contralateral hemisphere were also dissected and labeled as ACcont, ASCcont, PCcont, and PSCcont. Brain samples were frozen in liquid nitrogen then stored at -80°C until use. A 10% (w/v) tissue homogenate was mixed with 20 mmol/L potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide (Sigma), sonicated for 30 seconds, subjected to three freeze/thaw cycles, and then sonicated again for 30 seconds. The samples were centrifuged at 12,000 rpm, at 4°C for 20 minutes. The supernatant was assayed for myeloperoxidase activity.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Diagrammatic illustration of forebrain dissection into eight segments. (A) Anterior cortical ipsilateral (ACips), anterior subcortical ipsilateral (ASCips), anterior cortical contralateral (ACcontr), anterior subcortical contralateral (ASCcont). (B) Posterior cortical ipsilateral (PCips), posterior subcortical ipsilateral (PSCips), posterior cortical contralateral (PCcontr), posterior subcortical contralateral (PSCcont). The brain region histopathologically vulnerable to F-P-injury is shown in black.

Full figure and legend (248K)

MPO activity in supernatant was determined in 0.3 mL of 0.1 mol/L potassium phosphate buffer (pH 6.0) containing 1.25 mg/mL o-dianisidine and 0.05% H2O2. The assay was started by adding 30 to 40 muL of sample. A change in absorbance at 460 nm after 5 minutes was measured in an Elisa Plate Reader and the myeloperoxidase activity was calculated using a standard curve prepared for purified myeloperoxidase (Sigma). The linear portion of the absorbance at 460 nm was used to determine myeloperoxidase activity. One unit of myeloperoxidase activity was defined as that degrading 1 mumol of H2O2 in 1 minute. The myeloperoxidase enzymatic activity was proportional to the amount of sample assayed (20, 40, 80, and 100 muL) and was abolished by boiling the extract for 3 minutes. Data were expressed as units per gram of wet tissue, and an increase or decrease in enzymatic activity of ipsilateral or contralateral structures was compared with activities from sham-operated animals.

Immunohistochemical studies

At 3 hours, 1 day, 3 days, or 7 days after TBI or sham operation, a subset of rats under normothermic conditions were deeply anesthetized with halothane and perfused transcardially with 250 mL of cold physiologic saline for 1 minute and then FAM (a mixture of 40% formaldehyde, glacial acetic acid, and methanol; 1:1:8 by volume) for 20 minutes (25°C at a pressure of 100 mm Hg). Brains were extracted and stored in chilled fixative overnight. The next day, brains with the dura attached were embedded in paraffin and semiserial 10-mum sections were taken through the neuraxis. Sections were mounted on slides, rehydrated, and placed in 6% H2O2 to block endogenous peroxidase activity. The tissue was rinsed and steamed with a solution of citrate buffer for 20 minutes. Then the tissue was rinsed with 0.05 mol/L phosphate-buffered saline. Nonspecific activity was blocked with normal horse serum. Subsequently, brain sections were incubated at room temperature for 1 hour in a 1:1500 dilution of rabbit antihuman myeloperoxidase (DAKO Corporation, Carpitaria, CA, U.S.A.). To test for nonspecific staining, negative controls were conducted where the primary antibody was omitted during tissue processing. Further rinsing was done with phosphate-buffered saline and secondary antibody applied. The slides were processed using the LSAB staining kit (DAKO Corp., Carpitaria, CA, U.S.A.) per the manufacturer's instructions. myeloperoxidase staining was visualized with peroxidase, and diaminobenzidine was used as a chromogen to yield a brown reaction product. Slides were washed in 0.5% Triton X-100 followed by 1% cupric sulfate to further intensify staining. Counterstaining was done with hematoxylin, and tissue was then dehydrated and coverslipped.

Statistical analysis

Data were expressed as mean values plusminus SD. Two-way analysis of variance (ANOVA) for repeated measures was used to compare physiologic variables measured over time. Between-group comparisons of physiologic data and myeloperoxidase data sets were made using ANOVA one-way analysis of variance. Differences were considered significant if the P value was <0.05.

Top

RESULTS

Physiologic Parameters

Group 1. The physiologic variables obtained 5 minutes before and 15 minutes after TBI are presented in Table 1. No significant difference was observed between groups.


Groups 2 to 4 (3-hour survival). The physiologic variables measured 5 minutes before and 3 hours after TBI are presented in Table 2. During the 3 hour experimental period, brain temperatures differed significantly between normothermic (36.62 plusminus 0.172°C), hypothermic (30.18 plusminus 0.28°C), and hyperthermic (39.68 plusminus 0.44°C) groups (P < 0.05). PO2 differed at 15 minutes between normothermic versus hyperthermic rats (P < 0.05), but values were within normal ranges in both cases. There were no differences between the groups before TBI.


Groups 4 to 6 (3-day survival). The physiologic variables measured 5 minutes before and 15 minutes and 3 days after TBI are presented in Table 3. At 5, 15, minutes, and 3 hours after TBI brain temperatures differed significantly between the normothermic, hypothermic, and hyperthermic groups (P < 0.05). Baseline PCO2 differed between normothermic versus hypothermic rats (P < 0.05), and PO2 differed between normothermic versus hypothermic rats (P < 0.05) at 3 days after trauma. However, PCO2 and PO2 values were within normal ranges in all cases.


MPO activity after normothermic TBI

First phase of experiments. The time course of myeloperoxidase activity in the injured and noninjured cortex is illustrated in Fig. 2. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased (P < 0.05) at 3 hours within ipsilateral brain regions including the anterior (1.2 to -1.8 mm bregma) cortical segment (213.97 plusminus 56.2 versus control 65.5 plusminus 52.3 U/g of wet tissue; mean plusminus SD) and the posterior (-1.8 to -4.8 mm bregma, injured) cortical and subcortical segments compared to sham-operated rats (305.76 plusminus 27.8 and 258.67 plusminus 101.4 U/g of wet tissue versus control 62.8 plusminus 24.8 and 37.28 plusminus 35.6 U/g of wet tissue; mean plusminus SD, P < 0.0001, P < 0.05, respectively). At 24 hours after trauma only the injured cortical region (200.89 plusminus 57.7 versus control 62.8 plusminus 24.94 U/g of wet tissue; mean plusminus SD, P < 0.005) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was significantly increased within the ipsilateral anterior cortical segment (183.3 plusminus 101.1 versus control 65.5 plusminus 52.3 U/g of wet tissue; mean plusminus SD, P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex (619.68 plusminus 53.2 and 133.86 plusminus 57.22 U/g of wet tissue versus control 62.88 plusminus 24.94 and 37.28 plusminus 34.76 U/g of wet tissue, respectively; mean plusminus SD, P < 0.0001, P < 0.05). At 7 days there was a significant increase in myeloperoxidase activity only at the posterior cortical region compared to control (124.13 plusminus 48.02 versus 48.64 plusminus 24.8 U/g of wet tissue, respectively; mean plusminus SD, P < 0.05). No significant increase in myeloperoxidase activity was documented in the 24-hour sham-operated control brains.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Time course of neutrophil accumulation (myeloperoxidase activity) in injured cortical and subcortical brain regions and in respective contralateral regions or sham-operated animals. Within the ipsilateral cerebral cortex, a significant increase of myeloperoxidase activity was observed at 3 hours, 24 hours, 3 days, and 7 days after TBI (significantly different compared to sham-operated rats, *P < 0.05, **P < 0.005, ***P < 0.0001 versus control by one-way ANOVA). Values are the mean plusminus SD of n = 4 in each group.

Full figure and legend (83K)

Second phase of experiments. Figure 3 and Figure 4 depict the effects of posttraumatic hypothermia and hyperthermia on myeloperoxidase activity at 3 hours and 3 days after TBI. At both survival periods, posttraumatic hypothermia significantly reduced myeloperoxidase activity throughout the ipsilateral hemisphere including anterior and posterior cortical and subcortical structures compared to normothermic levels (P < 0.05). For example posttraumatic hypothermia significantly reduced myeloperoxidase activity in the injured posterior cortical region at 3 hours after TBI (47 plusminus 23.08 versus normothermic control 160.2 plusminus 44.6 U/g of wet tissue, n = 4). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior (injured) cortical region compared to normothermic values; 3 hours after TBI (473.5 plusminus 258.4 versus control 160.2 plusminus 44.66 U/g of wet tissue, n = 4, n = 5 respectively, P < 0.05) and 3 days after TBI (100.11 plusminus 27.58 versus 40.52 plusminus 21.8 U/g of wet tissue, mean plusminus SEM, n = 4, respectively, P < 0.05). In preliminary experiments we found that in normothermic TBI rats, extending the period of physiologic monitoring to 3 hours after TBI significantly reduced overall myeloperoxidase activity (Fig 3 and Fig 4). For this reason a separate set of normothermic rats was used in studies where 3 hours of posttraumatic monitoring was conducted. Although the mechanism for this consequence in myeloperoxidase activity was not investigated, the potential effects of prolonged anesthesia on myeloperoxidase activity should be considered.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Graph depicting myeloperoxidase activity at 3 hours in cortical and subcortical areas anterior and posterior to the histopathologically vulnerable region. *P < 0.05 for 30°C versus 37°C and 39°C versus 37°C. Brain temperature was controlled for 3 hours at 30°C, or 39°C after TBI (*P < 0.05 versus normothermic control by one-way ANOVA). Values are the mean plusminus SD of n = 4 to 5 in each group.

Full figure and legend (73K)

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Graph depicting myeloperoxidase activity at 3 days in cortical and subcortical areas anterior and posterior to the histopathologically vulnerable region. *P < 0.05 for 30°C versus 37°C and 39°C versus 37°C. Brain temperature was controlled for 3 hours at 30°C, 37°C, or 39°C after TBI. The rats were kept alive for 3 days and then killed after saline perfusion (*P < 0.05 versus normothermic control by one-way ANOVA). Values are the mean plusminus SD of n = 4 to 5 in each group.

Full figure and legend (78K)

Immunocytochemical findings

Immunocytochemical analysis at 3 hours, 24 hours, 3 days, and 7 days demonstrated large numbers of immunoreactive vascular and parenchymal leukocytes associated with normal appearing and damaged tissue (Fig. 5). At 24 hours after TBI, myeloperoxidase immunoreactive PMNLs were seen within subarachnoid spaces overlying the histopathologically vulnerable cortical region (Fig 5A and Fig 5B). In addition, PMNLs were associated with blood vessels within and adjacent to the contusion site. High-magnification analysis demonstrated both luminal and perivascular leukocytes (Fig 5C and Fig 5D). Scattered immunoreactive cells were observed within the damaged cortical tissue (Fig. 5E) and within the subarachnoid space (Fig. 5F). PMNLs were also identified within pial vessels, the interhemispheric fissure, hippocampus, and dentate gyrus. At 3 days immunoreactive leukocytes remained present within the histopathologically damaged cerebral cortex and contusion area. At 7 days PMNLs were observed within the injured cortical tissue and associated with newly formed vessels. In contrast, sham-operated control brain sections did not demonstrate abnormal neutrophil accumulation.

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Immunocytochemical visualization of neutrophils that exhibit myeloperoxidase immunoreactivity in ipsilateral brain regions after TBI. PMNLs are present in the subarachnoid space overlying the histopathologically vulnerable cortical region (A) and associated with pial vessels (B,*) at 24 hours after TBI. Within the contusion, vascular and perivascular PMNLs are observed (E, C, D). Scattered immunoreactive cells are also observed in cortical regions remote from the contused site as well as in areas showing histopathologic damage (D and E). High-magnification view (F) of cells exhibiting myeloperoxidase immunoreactivity within the subarachnoid space and superficial cortical layers (A, B, D, E, times 250; C, F, times 1,000).

Full figure and legend (512K)

Top

DISCUSSION

The results of the present study demonstrate that in this F-P model of brain injury neutrophils accumulate as part of the acute and subacute inflammatory response to injury. The time-course data show that posttraumatic cortical and subcortical PMNL accumulation, as measured by myeloperoxidase activity, was increased within 3 hours, peaked at 3 days, and resolved 7 days after trauma. We further demonstrated that posttraumatic temperature manipulations significantly influenced the degree of inflammation at both the acute and more chronic survival periods.

The accumulation of neutrophils observed at 3 hours after F-P may represent hemorrhage associated with vascular injury resulting from shear forces generated by the primary impact (Cortez et al., 1989; Dietrich et al., 1990a; Schoettle et al., 1990). We further demonstrated increased neutrophil accumulation at 24 to 72 hours with a peak of myeloperoxidase activity observed at 3 days after trauma. At 3 days increased myeloperoxidase activity was present in both vulnerable and nonvulnerable brain regions. These regionally specific alterations in myeloperoxidase activity at later time points could be attributable to widespread pathophysiologic events including subacute inflammatory responses, cerebrovascular disturbances, edema, and intracranial hypertension (Obrist et al., 1984; Shoettle et al., 1990; Biagas et al., 1992; Uhl et al., 1994). These findings are consistent with those reported in the F-P (Grady et al., 1999), in weight drop, and controlled cortical impact (CCI) models of TBI (Horner et al., 1992; Clark et al., 1994). In all these models, myeloperoxidase activity increased at 24 and 48 hours and resolved by 7 days after TBI. In addition, myeloperoxidase activity in the CCI model was reported to increase as a function of injury severity (Clark et al., 1996).

The time course of myeloperoxidase activity after TBI parallels traumatic events including alterations in local cerebral blood flow, cerebral edema, BBB damage, intracranial hypertension, and free radical formation (Chan et al., 1984; Obrist et al., 1984; Schoettle et al., 1990; Biagas et al., 1992; Uhl et al., 1994; Globus et al., 1995; Soares et al., 1995; Dietrich et al., 1996a). Previous studies by Bareyre et al. (1997) have reported the development of vasogenic edema resulting from BBB break-down caused by cortical vascular endothelial injury as early as 1 hour and as late as 5 days after TBI. Focal ischemia studies also demonstrated the myeloperoxidase activity to be significantly increased at 24 hours after MCAO (Bareyre et al., 1997). In addition, Clark and colleagues (1996) in a CCI model have shown that neutrophil accumulation occurs rapidly in ischemic regions. Thus, the degree of abnormal perfusion after injury may be an important factor for PMNL accumulation (Zhang et al., 1994a). The possibility that the alterations in myeloperoxidase activity are attributable to vasodilation/capillary recruitment or neovascularity at later time points should also be considered.

Trauma-induced alterations of neutrophils and macrophages/microglia may alter pathologic outcome through the secretion of proinflammatory cytokines including tumor necrosis factor-alpha, IL-1, IL-6, leukotrienes (Ellis et al., 1981; Goodman, 1986, McClain et al., 1991; Taupin et al., 1993; Shohami et al., 1994, 1996; Fan et al., 1995, 1996), complement, integrin (Kaczorowski et al., 1995), platelet activating factor (Patel et al., 1992), and platelet activating factor-induced active oxygen species (H2O2 and superoxide anion) (Arnould et al., 1993). When stimulated the activated PMNs consume oxygen (Nauseef et al., 1983) known as respiratory burst, phagocytose debris (Persson, 1976; Giulian et al., 1989) and release potent oxygen species that are antibacterial agents (Ward et al., 1988). The most important of these oxygen species are hydrogen peroxide (H2O2), the superoxide anion (O2-), hypochlorous acid, hydroxyl radicals, and nitric oxide (NO). However, a number of enzymes are also secreted by activated neutrophils, including superoxide dismutase that converts superoxide anion to H2O2 and myeloperoxidase which converts H2O2 to hypochlorous acid and oxygen-free radical (O-) (Zhang and Chopp, 1997). These reactive oxygen species can damage surrounding healthy nervous tissue by lipid peroxidation (Shappell and Smith, 1994; Anderson, 1995). Secreted factors may also lead to the upregulation of adhesion molecule expression by vascular endothelial cells which in turn attract circulating neutrophils. In this regard, selective therapeutic strategies targeted toward neutrophil depletion or blockers of PMNL adherence have been investigated in a number of cerebral ischemia studies (Sekiya et al., 1989; Biagas et al., 1992; Chen et al., 1994; Chopp et al., 1994; Uhl et al., 1994; Zhang et al., 1994b; McIntosh et al., 1996) and after F-P injury (Grady et al., 1999).

Previous studies have discussed the beneficial effects of mild and moderate hypothermia on secondary damage after TBI (Clifton et al., 1993; Marion et al., 1993; Palmer et al., 1993; Dietrich et al., 1994b; Chatzipanteli et al., 1999). The level of moderate hypothermia (30°C) used in our studies was similar to that used in previous experimental studies (Jiang et al., 1992; Lyeth et al., 1993; Dietrich et al., 1994b; Bramlett et al., 1995; Chatzipanteli et al., 1999). Other experimental studies have used more clinically relevant levels of mild hypothermia (Clark et al., 1996; Whalen et al., 1997a, 1997b). In clinical studies, mild hypothermia has also been used (Clifton et al., 1993; Marion et al., 1993; Clifton 1995; Marion and White, 1996). Possible hypothermic mechanisms underlying the beneficial effects include consequences on excitatory amino acids, metabolic changes, edema formation, BBB damage, local cerebral blood flow, marked reduction of IL-1 mRNA, and free-radical formation (Busto et al., 1989; Jiang et al., 1992; Clifton et al., 1993; Goss et al., 1995; Dietrich et al., 1996a; Toyoda et al., 1996). Previous studies have reported that posttraumatic hypothermia leads to reductions in cerebral blood flow (Marion et al., 1993; Marion and White, 1996; Zhao et al., 1999). The present findings emphasize the importance of posttraumatic brain temperature on inflammatory processes.

A study by Rosomoff et al. (1965) was the first to show the effects of hypothermia on the acute inflammatory response to experimental brain injury, where hypothermia (25°C) applied for 1 hour after injury delayed neutrophil accumulation up to 6 hours. Further studies have demonstrated that PMNL migration in vivo and in vitro was markedly reduced by profound hypothermia (29°C). In a focal ischemia-reperfusion model, hypothermia (30°C) reduced the cell-mediated inflammatory response (quantified by myeloperoxidase measurements) in the pericore region (Toyoda et al., 1996). Recent myeloperoxidase activity data and histologic outcome measurements have also demonstrated that mild hypothermia (32°C) reduced PMNL accumulation after CCI injury compared to hyperthermia (39°C) by fourfold using immunohistochemistry and by eightfold using the myeloperoxidase assay (Whalen et al., 1997a). Interestingly, E-selectin activation 4 hours after CCI was decreased in that study only modestly by hypothermia (32°C). It was concluded that neutrophil accumulation is dependent on posttraumatic brain temperature and that this effect is independent of the changes in the expression of E-selectin, ICAM-1, and absolute neutrophil count (Whalen et al., 1997a, 1997b).

In contrast to hypothermia, posttraumatic hyperthermia appears to aggravate the inflammatory consequences of TBI. Previous TBI studies reported that delayed posttraumatic hyperthermia (39°C) increased mortality rate, contusion volume, white matter pathology, and PMNL accumulation in the injured brain during the initial 4 hours after TBI (Dietrich et al., 1994b; Whalen et al., 1997a, 1997b). Furthermore, hyperthermia had no effect in the expression of E-selectin, ICAM-1, and absolute neutrophil count at this early time point after TBI (Whalen et al., 1997a, 1997b). The present findings therefore support previous observations and provide quantitative data indicating that hyperthermia increases the acute and more chronic inflammatory response to TBI. These temperature findings do not prove a cause-and-effect relationship between posttraumatic hyperthermia and injury outcome. Thus, it will be important in future studies to determine specific pathomechanisms underlying hyperthermia-induced aggravated PMNL accumulation.

In conclusion, these results indicate that posttraumatic inflammation plays an important role in the pathophysiology of moderate parasagittal F-P brain injury. We demonstrated a temporal response of myeloperoxidase activity in histopathologic vulnerable and nonvulnerable brain regions. In addition, treatment with therapeutic hypothermia significantly decreased myeloperoxidase activity whereas hyperthemia increased myeloperoxidase activity. Thus, a possible mechanism by which posttraumatic temperature modifications influence traumatic outcome is by altering the acute and more chronic inflammatory response to injury. A major objective in TBI research is to develop novel treatments that will prevent secondary tissue damage and promote neuronal survival. It is possible that combination therapy including mild hypothermia with pharmacologic agents that block PMNL accumulation may prove useful in the treatment of TBI.

Top

References

  1. Akriotis V & Biggar WD. (1985) The effects of hypothermia on neutrophil function in vitro. J Leukoc Biol 37: 51−61. | PubMed | ChemPort |
  2. Albelda SM, Smith CW & Ward PA. (1994) Adhesion molecules and inflammatory injury. FASEB J 8: 504−512. | PubMed | ISI | ChemPort |
  3. Anderson R. (1995) The activated neutrophil-formidable forces unleased. S Afr Med J 85: 1024−1028. | PubMed | ChemPort |
  4. Arnould T, Michiels C & Remacle J. (1993) Increased PMN adherence on endothelial cells after hypoxia: involvement of PAF, CD18/CD11b, and ICAM-1. Am J Physiol 264: C1102−1110. | PubMed | ISI | ChemPort |
  5. Bareyre F, Wahl F, McIntosh TK & Stutzmann JM. (1997) Time course of cerebral edema after traumatic brain injury in rats: effects of riluzole and mannitol. J Neurotrauma 14: 839−849. | PubMed | ChemPort |
  6. Barone FC, Hillegass LM, Price WJ, White RF, Lee EV, Feuerstein GZ, Sarau HM, Clark RK & Griswold DEJ. (1991) Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histologic verification. Neurosci Res 29: 336−345. | ChemPort |
  7. Beynon HL, Haskard DO, Davies KA, Haroutunian R & Walport MJ. (1993) Combinations of low concentrations of cytokines and acute agonists synergize in increasing the permeability of endothelial monolayers. Clin Exp Immunol 91: 314−319. | PubMed | ChemPort |
  8. Biagas KV, Uhl MW, Schiding JK, Nemoto EM & Kochanek PM. (1992) Assessment of posttraumatic polymorphonuclear leukocyte accumulation in rat brain using tissue myeloperoxidase assay and vinblastine treatment. J Neurotrauma 9: 363−71. | PubMed | ChemPort |
  9. Biggar WD, Bohn DJ, Kent G, Barker C & Hamilton G. (1984) Neutrophil migration in vitro and in vivo during hypothermia. Infect Immun 46: 857−859. | PubMed | ChemPort |
  10. Bramlett H, Green EJ, Dietrich WD, Busto R, Globus MY-T & Ginsberg MD. (1995) Posttraumatic brain hypothermia provides protection from sensorimotor and cognitive behavioral deficits. J Neurotrauma 12: 289−298. | PubMed | ChemPort |
  11. Busto R, Globus MYT, Dietrich WD, Martinez E, Valdes I & Ginsberg MD. (1989) Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain. Stroke 20: 904−910. | PubMed | ChemPort |
  12. Carlos TM, Clark RS, Franicola-Higgins D, Schiding JK & Kochanek PM. (1997) Expression of endothelial adhesion molecules and recruitment of neutrophils after traumatic brain injury in rats. J Leukoc Biol 61: 279−285. | PubMed | ChemPort |
  13. Carlos TM & Harlan JM. (1994) Leukocyte-endothelial adhesion molecules. Blood 84: 2068−2101. | PubMed | ISI | ChemPort |
  14. Chan PH, Schmidley JW, Fishman RA & Longar SM. (1984) Brain injury, edema, and vascular permeability changes induced by oxygen-derived free radicals. Neurology 34: 315−320. | PubMed | ChemPort |
  15. Chatzipanteli K, Wada K, Busto R & Dietrich WD. (1999) The effects of moderate hypothermia on constitutive and inducible nitric oxide synthase activities following traumatic brain injury in the rat. J Neurochem 72: 2047−2052. | Article | PubMed | ChemPort |
  16. Chen H, Chopp M & Welch KM. (1991) Effect of mild hyperthermia on the ischemic infarct volume after middle cerebral artery occlusion in the rat. Neurology 41: 1133−1135. | PubMed | ChemPort |
  17. Chopp M, Zhang RL, Chen H, Li Y, Jiang N & Rusche JR. (1994) Postischemic administration of an anti-Mac-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in rats. Stroke 25: 869−875. | PubMed | ISI | ChemPort |
  18. Clark RS, Kochanek PM, Marion DW, Schiding JK, White M, Palmer AM & DeKosky ST. (1996) Mild postraumatic hypothermia reduces mortality after severe controlled cortical impact in rats. J Cereb Blood Flow Metab 16: 253−261. | PubMed | ChemPort |
  19. Clark RS, Schiding JK, Kaczorowski SL, Marion DW & Kochanek PM. (1994) Neutrophil accumulation after traumatic brain injury in rats: comparison of weight drop and controlled cortical impact models. J Neurotrauma 11: 499−506. | PubMed | ChemPort |
  20. Clifton GL. (1995) Systemic hypothermia in treatment of severe brain injury: a review and update. J Neurotrauma 12: 923−927. | PubMed | ChemPort |
  21. Clifton GL, Jiang JY, Lyeth BG, Jenkins LW, Hamm RJ & Hayes RL. (1993) Marked protection by moderate hypothermia after experimental traumatic brain injury. J Cereb Blood Flow Metab 11: 114−121.
  22. Cortez S, McINtosh TK & Noble LJ. (1989) Experimental fluid percussion brain injury: vascular disruption and neuronal glial alterations. Brain Res 482: 271−282. | Article | PubMed | ChemPort |
  23. David S, Bouchard G, Tsatas O & Giftochristos N. (1990) Macrophages can modify the nonpermissive nature of adult mammalian centrous system. Neuron 5: 463−469. | Article | PubMed | ChemPort |
  24. Del Zoppo GJ & Garcia JH. 1995 Polymorphonuclear leukocyte adhesion in cerebrovascular ischemia: pathophysiologic implications of leukocyte adhesion. (In) Physiology and Pathophysiology of Leukocyte Adhesion (Granger DN, Semid-Schonbein GW, eds) New York: Oxford University Press (pp) 408−425. | ChemPort |
  25. Dietrich WD, Alonso O, Busto R & Ginsberg MD. (1994b) Posttraumatic brain hypothermia reduces histopathological damage following concussive brain injury in the rat. Acta Neuropathol 87: 250−258. | Article | PubMed | ChemPort |
  26. Dietrich WD, Alonso O, Busto R, Prado R, Dewanjee S, Dewanjee S, Dewanjee MK & Ginsberg MD. (1996a) Widespread hemodynamic depression and focal platelet accumulation after fluid percussion brain injury: a double-label autoradiographic study in rats. J Cereb Blood Flow Metab 16: 481−489. | ChemPort |
  27. Dietrich WD, Alonso O & Halley M. (1994a) Early microvascular and neuronal consequences of traumatic brain injury: a light and electron microscopic study in rats. J Neurotrauma 11: 289−301. | PubMed | ChemPort |
  28. Dietrich WD, Alonso O, Halley M & Busto R. (1996b) Delayed posttraumatic brain hyperthermia worsens outcome after fluid percussion brain injury: a light and electron microscopic study in rats. Neurosurgery 38: 533−541. | Article | PubMed | ChemPort |
  29. Dietrich WD, Busto R, Globus MY & Ginsberg MD. (1996c) Brain damage and temperature: cellular and molecular mechanisms. Adv Neurol 71: 177−794. | PubMed | ChemPort |
  30. Dietrich WD, Busto R, Valdes I & Halley M. (1990b) The importance of brain temperature in alterations of the blood-brain barrier during brain ischemia. J Neuropathol Exp Neurol 49: 486−497. | PubMed | ISI | ChemPort |
  31. Dietrich WD, Busto R, Valdes I & Loor Y. (1990a) Effects of normothermic versus mild hyperthermic forebrain ischemia in rats. Stroke 21: 1318−1325. | PubMed | ChemPort |
  32. Dixon CE, Lyeth BG, Povlishock JT, Findling RL, Hamm RJ, Marmarou A, Young HF & Hayes RL. (1987) A fluid percussion model of experimental brain injury in the rat. J Neurosurg 67: 110−119. | PubMed | ChemPort |
  33. Ellis EF, Wright KF, Wei EP & Kontos HA. (1981) Cyclooxygenase products of arachidonic acid metabolism in cat cerebral cortex after experimental concussive brain injury. J Neurochem 37: 892−896. | PubMed | ChemPort |
  34. Fan L, Young PR, Barone FC, Feuerstein GZ, Smith DH & McIntosh TK. (1996) Experimental brain injury induces differential expression of tumor necrosis factor-alpha mRNA in the CNS. Brain Res Mol Brain Res 36: 287−291. | PubMed | ChemPort |
  35. Fan L, Young PR, Barone FC, Feuerstein GZ, Smith DH & McIntosh TK. (1995) Experimental brain injury induces expression of interleukin-1 beta mRNA in the rat brain. Brain Res Mol Brain Res 30: 125−130. | Article | PubMed | ChemPort |
  36. Giulian D, Chen J, Ingeman JE, George JK & Noponen M. (1989) The role of mononuclear phagocytes in wound healing after traumatic injury to adult mammalian brain. J Neurosci 9: 4416−4429. | PubMed | ChemPort |
  37. Globus MY, Alonso O, Dietrich WD, Busto R & Ginsberg MD. (1995) Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia. J Neurochem 65: 1704−1711. | PubMed | ChemPort |
  38. Goodman MG. (1986) Regulation of B cell activation by prostaglandins: cell cycle-specific effects on activation by anti-immunoglobulin and 8-mercaptoguanosine. J Immunol 15: 3753−3777.
  39. Goss JR, Styren SD, Miller PD, Kochanek PM, Palmer AM, Marion DW & DeKosky ST. (1995) Hypothermia attenuates the normal increase in interleukin 1 beta RNA and nerve growth factor following traumatic brain injury in the rat. J Neurotrauma 12: 159−167. | PubMed | ChemPort |
  40. Grady MS, Cody RF, Maris DO, McCall TD, Seckin H, Sharar SR & Winn HR. (1999) P-Selectin blockade following fluid-percussion injury: behavioral and immunochemical sequelae. J Neurotrauma 16: 13−25. | PubMed | ChemPort |
  41. Grogaard B, Schurer L, Gerdin B & Arfors KE. (1989) Delayed hypoperfusion after incomplete forebrain ischemia in the rat. The role of polymorphonuclear leukocytes. J Cereb Blood Flow Metab 9: 500−505. | PubMed | ChemPort |
  42. Hallenbeck JM, Dutka AJ, Tanishima T, Kochanek PM, Kumaroo KK, Thompson CB, Obrenovitch TP & Contreras TJ. (1986) Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. Stroke 17: 246−245. | PubMed | ChemPort |
  43. Hama T, Miyamoto M, Tsukui H, Nishio C & Hatanaka H. (1989) Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats. Neurosci Lett 104: 340−344. | Article | PubMed | ChemPort |
  44. Hamada Y, Ikata T, Katoh S, Nakauchi K, Niwa M, Kawai Y & Fukuzawa K. (1996) Involvement of an intercellular adhesion molecule 1-dependent pathway in the pathogenesis of secondary changes after spinal cord injury in rats. J Neurochem 66: 1525−1531. | PubMed | ChemPort |
  45. Hartl R, Medary MB, Ruge M, Arfors KE & Ghajar J. (1997) Early white blood cell dynamics after traumatic brain injury: effects on the cerebral microcirculation. J Cereb Blood Flow Metab 17: 1210−1220. | PubMed | ChemPort |
  46. Horner HC, Kraemer M, Schroeter M, Witte OW & Stoll G. (1992) Characterization of leukocyte infiltration in traumatic brain injury in the rat. Soc Neurosci Abstr 18: 173.
  47. Jean WC, Spellman SR, Nussbaum ES & Low WC. (1998) Reperfusion after focal cerebral ischemia: the role of inflammation and the therapeutic horizon. Neurosurgery 43: 1382−1396. | PubMed | ChemPort |
  48. Jiang JY, Lyeth BG, Clifton GL, Jenkins LW, Hamm RJ & Hayes RL. (1991) Relationship between body and brain temperature in traumatically brain-injured rodents. J Neurosurg 74: 492−496. | PubMed | ChemPort |
  49. Jiang JY, Lyeth BG, Kapasi MZ, Jenkins LW & Povlishock JT. (1992) Moderate hypothermia reduces blood-brain barrier disruption following traumatic brain injury in the rat. Acta Neuropathol (Berl) 84: 495−500. | PubMed | ChemPort |
  50. Kaczorowski SL, Schiding JK, Toth CA, Kochanek PM, Kaczorowski SL, Schiding JK, Toth CA & Kochanek PM. (1995) Effect of soluble complement receptor-1 on neutrophil accumulation after traumatic brain injury in rats. J Cereb Blood Flow Metab 15: 860−864. | PubMed | ISI | ChemPort |
  51. Kochanek PM, Dutka AJ, Kumaroo KK & Hallenbeck JM. (1987) Platelet activating factor receptor blockade enhances recovery after multifocal brain ischemia. Life Sci 41: 2639−2644. | Article | PubMed | ChemPort |
  52. Kochanek PM & Hallenbeck JM. (1992) Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 23: 1367−1379. | PubMed | ISI | ChemPort |
  53. Lindholm D, Heumann R, Meyer M & Thoenen H. (1987) Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature 330: 658−659. | Article | PubMed | ChemPort |
  54. Lyeth BG, Jiang JY, Robinson SE, Guo H & Jenkins LW. (1993) Behavioral protection by moderate hypothermia initiated after experimental traumatic brain injury. J Neurotrauma 10: 57−64. | PubMed | ChemPort |
  55. Mackay CR & Imhof BA. (1993) Cell adhesion in the immune system. Immunol Today 14: 99−102. | Article | PubMed | ChemPort |
  56. Mantovani AF, Bussolino F & Introna M. (1997) Cytokine regulation of endothelial cell function: from molecular level to bedside. Immunol Today 18: 231−240. | Article | PubMed | ISI | ChemPort |
  57. Marion DW, Obrist WD, Carlier PM, Penrod LE & Darby JM. (1993) The use of moderate therapeutic hypothermia for patients with severe head injuries: a preliminary report. J Neurosurg 79: 354−362. | PubMed | ChemPort |
  58. Marion DW, Penrod LE, Kelsey MD, Obsrist WD, Kochanek PM, Palmer AM, Wiesniewski SR & DeKosky ST. (1997) Treatment of traumatic brain injury with moderate hypothermia. N Engl J Med 336: 540−546. | Article | PubMed | ChemPort |
  59. Marion DW & White MJ. (1996) Treatment of traumatic brain injury with moderate hypothermia and 21-aminosteroids. J Neurotrauma 13: 139−147. | PubMed | ChemPort |
  60. Matsuo Y, Kihara T, Ikeda M, Ninomiya M, Onodera H & Kogure K. (1995) Role of neutrophils in radical production during ischemia and reperfusion of the rat brain: effect of neutrophil depletion on extracellular ascorbyl radical formation. J Cereb Blood Flow Metab 15: 941−947. | PubMed | ChemPort |
  61. Matsuo Y, Onodera H, Shiga Y, Nakamura M, Ninomiya M, Kihara T & Kogure K. (1994a) Correlation between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in the rat. Effects of neutrophil depletion. Stroke 7: 1469−1475.
  62. Matsuo Y, Onodera H, Shiga Y, Shozuhara H, Ninomiya M, Kihara T, Tamatani T, Miyasaka M & Kogure K. (1994b) Role of cell adhesion molecules in brain injury after transient middle cerebral artery occlusion in the rat. Brain Res 656: 344−352. | Article | PubMed | ChemPort |
  63. McClain C, Cohen D, Phillips R, Ott L & Young B. (1991) Increased plasma and ventricular fluid interleukin 6 levels in patients with head injury. J Lab Clin Med 118: 225−231. | PubMed | ChemPort |
  64. McIntosh TK, Smith DH & Garde E. (1996) Therapeutic approaches for the prevention of secondary brain injury. Eur J Anaesthesiol 13: 291−309. | Article | PubMed | ChemPort |
  65. McIntosh TK, Vink R, Noble L, Yamakami I, Fernyak S, Soares H & Faden AL. (1989) Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model. Neuroscience 28: 233−244. | Article | PubMed | ChemPort |
  66. Morikawa E, Ginsberg MD, Dietrich WD, Duncan RC, Kraydieh S, Globus MY & Busto R. (1992) The significance of brain temperature in focal cerebral ischemia: histopathological consequences of middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 12: 380−389. | PubMed | ChemPort |
  67. Nauseef WM, Root RK & Malech HL. (1983) Biochemical and immunologic analysis of hereditary myeloperoxidase deficiency. J Clin Invest 71: 1297−1307. | PubMed | ChemPort |
  68. Obrist WD, Langfitt TW, Jaggi JL, Cruz J & Gennarelli TA. (1984) Cerebral blood flow and metabolism in comatose patients with acute head injury. Relationship to intracranial hypertension. J Neurosurg 61: 241−253. | PubMed | ChemPort |
  69. Ott L, McClain CJ, Gillespie M & Young B. (1994) Cytokines and metabolic dysfunction after severe head injury. J Neurotrauma 11: 447−472. | PubMed | ChemPort |
  70. Palmer AM, Marion DW, Botscheller ML & Redd EE. (1993) Therapeutic hypothermia is cytoprotective without attenuating the traumatic brain injury-induced elevations in interstitial concentrations of aspartate and glutamate. J Neurotrauma 10: 363−372. | PubMed | ChemPort |
  71. Patel KD, Zimmerman GA, Prescott SM & McIntyre TM. (1992) Novel leukocyte agonists are released by endothelial cells exposed to peroxide. J Biol Chem 267: 15168−15175. | PubMed | ChemPort |
  72. Persson L. (1976) Cellular reactions to small cerebral stab wounds in the frontal lobe. Virchows Arch B Cell Pathol 22: 21−37. | PubMed | ChemPort |
  73. Petito CK. (1979) Early and late mechanisms of increased vascular permeability following experimental cerebral infarction. J Neuropathol Exp Neurol 38: 222−234. | PubMed | ChemPort |
  74. Rosomoff HL, Clasen RA, Hartstock R & Bebin J. (1965) Brain reaction to experimental injury after hypothermia. Arch Neurol 13: 337−345. | PubMed | ChemPort |
  75. Rosomoff HL & Haladay DA. (1954) Cerebral blood flow and oxygen consumption during hypothermia. Am J Physiol 179: 85−88. | PubMed | ChemPort |
  76. Rothwell N, Allan S & Toulmond S. (1997) The role of interleukin 1 in acute neurodegeneration and stroke: pathophysiological and therapeutic implications. J Clin Invest 100: 2648−2652. | PubMed | ISI | ChemPort |
  77. Saito K, Levine L & Moskowitz MA. (1988) Blood components contribute to rise in gerbil brain levels of leukotriene-like immunoreactivity after ischemia and reperfusion. Stroke 19: 1395−1398. | PubMed | ChemPort |
  78. Schoettle RJ, Kochanek PM, Magargee MJ, Uhl MW & Nemoto EM. (1990) Early polymorphonuclear leukocyte accumulation correlates with the development of posttraumatic cerebral edema in rats. J Neurotrauma 7: 207−217. | PubMed | ChemPort |
  79. Sekiya S, Gotoh S, Yamashita T, Watanabi T, Saitoh S & Sendo F. (1989) Selective depletion of rat neutrophils by in vivo administration of monoclonal antibody. J Leukoc Biol 46: 96−102. | PubMed | ChemPort |
  80. Shappell SB & Smith CW. 1994 Acute inflammatory response: granulocyte migration and activation. (In) The Handbook of Immunopharmacology, Adhesion Molecules (Wegner CS, ed) Abbott Park, IL: Academic Press (pp) 29−70. | ChemPort |
  81. Shohami E, Bass R, Wallach D, Yamin A & Gallily R. (1996) Inhibition of tumor necrosis factor alpha (TNFalpha) activity in rat brain is associated with cerebroprotection after closed head injury. J Cereb Blood Flow Metab 16: 378−384. | PubMed | ChemPort |
  82. Shohami E, Gallily R, Mechoulam R, Bass R & Ben-Hur T. (1997) Cytokine production in the brain following closed head injury: dexanabinol (HU-211) is a novel TNF-alpha inhibitor and an effective neuroprotectant. J Neuroimmunol 72: 169−177. | Article | PubMed | ChemPort |
  83. Shohami E, Novikov M, Bass R, Yamin A & Gallily R. (1994) Closed head injury triggers early production of TNF alpha and IL-6 by brain tissue. J Cereb Blood Flow Metab 14: 615−619. | PubMed | ChemPort |
  84. Smith CW. (1993) Leukocyte-endothelial cell interactions. Semin Hematol 30: 45−53. | PubMed | ChemPort |
  85. Soares HD, Hicks RR, Smith D & McIntosh TKJ. (1995) Inflammatory leukocytic recruitment and diffuse neuronal degeneration are separate pathological processes resulting from traumatic brain injury. J Neurosci 15: 8223−8233. | PubMed | ChemPort |
  86. Stoolman LM. (1993) Adhesion molecules involved in leukocyte recruitment and lymphocyte recirculation. Chest 103: 79S−86. | PubMed | ChemPort |
  87. Taft WC, Yang K, Dixon CE, Clifton GL & Hayes RL. (1993) Hypothermia attenuates the loss of hippocampal microtubule-associated protein 2 (MAP2) following traumatic brain injury. J Cereb Blood Flow Metab 13: 796−802. | PubMed | ChemPort |
  88. Taupin V, Toulmond S, Serraro A, Benavides J & Zavala F. (1993) Increase in IL-6, IL-1, and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro54864, a peripheral type (p site) benzodiazepine ligand. J Neuroimmunol 42: 177−185. | Article | PubMed | ISI | ChemPort |
  89. Tomina M & Fukuuchi Y. (1996) Leukocyte, macrophages and secondary brain damage following cerebral ischemia. Acta Neurochir Suppl (Wien) 66: 32−39. | ChemPort |
  90. Toyoda T, Suzuki S, Kassell NF & Lee KS. (1996) Intraischemic hypothermia attenuates neutrophil infiltration in the rat neocortex after focal ischemia-reperfusion injury. Neurosurgery 39: 1200−1205. | PubMed | ChemPort |
  91. Uhl MW, Biagas KV, Grundl PD, Barmada MA, Schiding JK, Nemoto EM & Kochanek PM. (1994) Effects of neutropenia on edema, histology, and cerebral blood flow after traumatic brain injury in rats. J Neurotrauma 3: 303−315.
  92. Wahl M, Unterberg A, Baethmann A & Schilling L. (1988) Mediators of blood-brain barrier dysfunction and formation of vasogenic brain edema. J Cereb Blood Flow Metabol 8: 621−634. | ChemPort |
  93. Ward PA, Warren JS & Johnson KJ. (1988) Oxygen radicals, inflammation, and tissue injury. Free Radic Biol Med 5: 403−408. | Article | PubMed | ChemPort |
  94. Whalen MJ, Carlos TM, Clark RS, Marion DW, DeKosky ST, Heineman S, Schiding JK, Memarzadeh F, Dixon CE & Kochanek PM. (1997b) The relationship between brain temperature and neutrophil accumulation after traumatic brain injury in rats. Acta Neurochir 70: 260−261. | ChemPort |
  95. Whalen MJ, Carlos TM, Clark RS, Marion DW, DeKosky ST, Heineman S, Schiding JK, Memarzadeh F & Kochanek PM. (1997a) The effect of brain temperature on acute inflammation after traumatic brain injury in rats. J Neurotrauma 14: 561−572. | PubMed | ChemPort |
  96. Whalen MJ, Carlos TM, Dixon CE, Schiding JK, Clark RS, Baum E, Yan HQ, Marion DW & Kochanek PM. (1999) Effect of traumatic brain injury in mice deficient in intercellular adhesion molecule-1: assessment of histopathologic and functional outcome. J Neurotrauma 16: 299−309. | PubMed | ChemPort |
  97. Zhang Gang Zhang MD & Chopp M. (1997) Measurement of myeloperoxidase immunoreactive cells in ischemic brain after transient middle cerebral artery occlusion in the rat. Neurosci Res Commun 20: 85−91. | Article | ChemPort |
  98. Zhang RL, Chopp M, Chen H & Garcia JH. (1994a) Temporal profile of ischemic tissue damage, neutrophil response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat. J Neurol Sci 125: 3−10. | Article | PubMed | ChemPort |
  99. Zhang RL, Chopp M, Li Y, Zaloga C, Jiang N, Jones ML, Miyasaka M & Ward PA. (1994b) Anti-ICAM-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in the rat. Neurology 44: 1747−1751. | PubMed | ISI | ChemPort |
  100. Zhao W, Alonso OF, Loor JY, Busto R & Ginsberg MD. (1999) Influence of early posttraumatic hypothermia therapy on local cerebral blood flow and glucose metabolism after fluid-percussion brain injury. J Neurosurg 90: 510−519. | PubMed | ChemPort |
  101. Zilles L. 1985 The Cortex of the Brain: A Stereotaxic Atlas Berlin: Springer-Verlag.
  102. Zimmerman J. 1986 Polymorphonuclear leukocytes-agents of host defence and autoinjury. (In) Critical Care State of the Art (Chernow B, Shoemaker S, eds) Fullerton, CA: Society of Critical Care Medicine (pp) 281−299.

Extra navigation

.

naturejobs

  • Postdoctoral Position

    • McGill University
    • Goodman Cancer Centre, McGill University, Cancer Pavilion, 1160 Pine Avenue West, Room 414, Montreal, Quebec , Canada, H3A 1A3

  • Behavioural Pharmacologist

    • Eisai London Research Laboratories Ltd
    • Hatfield, United Kingdom
More science jobs
Post a job for free

natureproducts


ADVERTISEMENT