Joanne K. Schiding

Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice

Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1.5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS–/–) were compared with wild-type (iNOS+/+) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS+/+ and iNOS–/– mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS–/– mice showed markedly worse performance in the MWM task than iNOS+/+ mice. A beneficial role for iNOS in TBI is supported.

Expression of endothelial adhesion molecules and recruitment of neutrophils after traumatic brain injury in rats.

Traumatic brain injury (TBI) is often accompanied by an acute inflammatory reaction mediated initially by neutrophils. Adhesion molecules expressed on vascular endothelium are requisite elements during recruitment of leukocytes at sites of inflammation. In a rat model of TBI the induction and persistent expression of E‐selectin (CD62E) on cerebrovascular endothelium ipsilateral, but not contralateral, to the site of contusion was demonstrated (P < 0.05 at 4 and 48 h posttrauma). In addition, these studies confirmed up‐regulation and prolonged expression of ICAM‐1 (CD54) on endothelium in the traumatized hemisphere (P < 0.05 at 4, 24, 48, and 72 h posttrauma). It is of interest that increased expression of CD54 was noted on blood vessels in the contralateral, non‐traumatized hemisphere 48 h posttrauma. Expression of a third endothelial adhesion molecule, PECAM‐1 (CD31), was unchanged following trauma. Administration of a murine monoclonal antibody (TM‐8) that inhibits the adhesive function of CD54 blocked a significant portion (37.9%) of neutrophil recruitment 24 h posttrauma (P = 0.04). Employing immunocytochemistry and a monoclonal antibody specific for rat neutrophils (RP‐3), peak infiltration of neutrophils was shown to occur 48 h after trauma. In contrast to emigration of neutrophils from blood vessels within the contusion, however, entry of neutrophils occurred from the surrounding leptomeninges and choroidal vessels. These studies demonstrate the relevance of CD54 (ICAM‐1) in recruitment of neutrophils following TBI. However, the majority of neutrophil influx relies on endothelial adhesion molecules other than CD54. Because emigration of neutrophils was shown to occur predominantly from vessels within the leptomeninges and choroid plexus, intrathecal delivery of agents that inhibit the adhesive interactions between neutrophils, endothelial CD54, and other endothelial adhesion molecules to be defined may offer a novel form of therapy to prevent the acute inflammatory response that follows TBI. J. Leukoc. Biol. 61: 279–285; 1997.

Inducible nitric oxide synthase expression in cerebrovascular smooth muscle and neutrophils after traumatic brain injury in immature rats

The inflammatory response after traumatic brain injury (TBI) includes cytokine production, leukocyte infiltration, and microglial activation. Production of nitric oxide by inducible nitric oxide synthase (iNOS) occurs during acute inflammation outside of the CNS and in models of cerebral ischemia, and therefore may contribute to the inflammatory response after TBI. The purpose of this study was to localize and define the time course of iNOS expression after TBI in the immature rat. Immature Wistar rats (age 3.5-4.5 wk) were anesthetized and subjected to percussive trauma to the right parietal cortex. Nontraumatized rats were used as controls (n = 7). At 2, 24, 48, or 168 h (n = 3/group) posttrauma rats were killed by perfusion fixation. Brains were removed, frozen, sectioned, immunostained with antibodies against iNOS and glial fibrillary acidic protein (GFAP, a marker specific for astrocytes), and imaged using fluorescent detection systems. There was no detectable expression of iNOS in control brains. At 2 h, minimal cerebrovascular iNOS expression was seen in the peritrauma area. At 24 and 48 h, there was marked peritrauma cerebrovascular iNOS expression that appeared to be restricted to vascular smooth muscle cells and infiltrated leukocytes. Further dual-immunolabeling showed that the leukocytes expressing iNOS were predominantly neutrophils. At 168 h, iNOS expression was no longer detectable. iNOS was not detectable in GFAP-positive cells. The prominent expression of iNOS protein after TBI in cerebrovascular smooth muscle cells and infiltrated neutrophils suggests that iNOS may play a role in cerebrovascular disturbances and secondary brain injury after trauma.

Mild Posttraumatic Hypothermia Reduces Mortality after Severe Controlled Cortical Impact in Rats

The effect of posttraumatic hypothermia (brain temperature controlled at 32°C for 4 h) on mortality after severe controlled cortical impact (CCI) was studied in rats. Four posttraumatic brain temperatures were compared: 37°C (n = 10), 36°C (n = 4), 32°C (n = 10), and uncontrolled (UC; n = 6). Rats were anesthetized and subjected to severe CCI (4.0-m/s velocity, 3.0-mm depth) to the exposed left parietal cortex. At 10 min posttrauma the rats were cooled or maintained at their target brain temperature, using external cooling or warming. Brain temperature in the UC group was recorded but not regulated, and rectal temperature was maintained at 37 ± 0.5°C. After 4 h, rats were rewarmed over a 1-h period to 37°C, extubated, and observed for 24 h. In the 37 and 36°C groups, 24-h mortality was 50% (37°C = 5/10, 36°C = 2/4). In the 32°C group, 24-h mortality was 10% (1/10). In the UC group, brain temperature was 35.4 ± 0.6°C during the 4-h treatment period and 24-h mortality was 0% (0/6). Mortality was higher in groups with brain temperatures ≥36°C versus those with brain temperatures <36°C (50 vs. 6%, respectively; p < 0.05). Additionally, electroencephalograms (EEG) were recorded in subsets of each temperature group and the percentage of time that the EEG was suppressed (isoelectric) was determined. Percentage of EEG suppression was greater in the hypothermic (32°C, n = 6; UC, n = 4) groups than in the normothermic(36°C, n = 3; 37°C, n = 6) groups (23.3 ± 14.3 vs. 1.2 ± 3.1%, respectively; p < 0.05). Posttraumatic hypothermia suppressed EEG during treatment and reduced mortality after severe CCI. The threshold for this protective effect appears to be a brain temperature <36°C. Thus, even mild hypothermia may be beneficial after severe brain trauma.

Cerebral perfusion during anesthesia with fentanyl, isoflurane, or pentobarbital in normal rats studied by arterial spin-labeled MRI

The influence of anesthetic agents on cerebral blood flow (CBF) was tested in normal rats. CBF is quantified with arterial spin‐labeled MRI in rats anesthetized with either an opiate (fentanyl), a potent inhalation anesthetic agent (isoflurane), or a barbiturate (pentobarbital) using doses commonly employed in experimental paradigms. CBF values were found to be about 2.5–3 times lower in most regions analyzed during anesthesia with either fentanyl (with N2O/O2) or pentobarbital vs. isoflurane (with N2O/O2), in agreement with findings utilizing invasive measurement techniques. CBF was heterogeneous in rats anesthetized with isoflurane (with N2O/O2), but relatively homogeneous in rats anesthetized with either fentanyl (with N2O/O2) or pentobarbital, also in agreement with studies using other techniques. Magn Reson Med 46:202–206, 2001.

Effect of Soluble Complement Receptor-1 on Neutrophil Accumulation after Traumatic Brain Injury in Rats

As part of the acute inflammatory response, neutrophils accumulate in the central nervous system after injury. Recently, a soluble human recombinant complement receptor (sCR1; BRL 55730; T Cell Sciences, Inc., Cambridge, MA, U.S.A.) has been developed that inhibits the activation of both the classical and the alternative pathways of complement. sCR1 attenuates the effects of the acute inflammatory response in several models of injury outside the central nervous system. The role of complement in traumatic brain injury, however, remains undefined. We hypothesized that treatment with sCR1 would attenuate neutrophil accumulation in the brain after cerebral trauma. Using a randomized, blinded protocol, 18 anesthetized Sprague–Dawley rats were pretreated with sCR1 or saline (control) at both 2 h and 2 min before trauma (weight drop) to the exposed right parietal cortex. A third dose of sCR1 (or saline) was given 6 h after trauma. Coronal brain sections centered on the site of trauma were obtained at 24 h after trauma and analyzed for myeloperoxidase (MPO) activity as a marker of neutrophil accumulation. Complete blood counts with differential were obtained before treatment with sCR1 and at 24 h after trauma. At 24 h after trauma, brain MPO activity was reduced by 41% in sCR1-treated rats compared with control rats [0.1599 ± 0.102 versus 0.27(2 ± 0.178 U/g (mean ± SD); p = 0.02]. The neutrophil count in peripheral blood increased approximately twofold in both groups. Neutrophil accumulation occurring in the brain after trauma is inhibited by sCR1 treatment. This suggests that complement activation is involved in the local inflammatory response to traumatic brain injury and plays an important role in neutrophil accumulation in the injured brain.

Assessment of Cerebral Blood Flow and CO2 Reactivity After Controlled Cortical Impact By Perfusion Magnetic Resonance Imaging Using Arterial Spin-Labeling in Rats

We measured CBF and CO2 reactivity after traumatic brain injury (TBI) produced by controlled cortical impact (CCI) using magnetic resonance imaging (MRI) and spin-labeled carotid artery water protons as an endogenous tracer. Fourteen Sprague-Dawley rats divided into TBI (CCI; 4.02 ± 0.14 m/s velocity; 2.5 mm deformation), sham, and control groups were studied 24 hours after TBI or surgery. Perfusion maps were generated during normocarbia (Paco2 30 to 40 mm Hg) and hypocarbia (Paco2 15 to 25 mm Hg). During normocarbia, CBF was reduced within a cortical region of interest (ROI, injured versus contralateral) after TBI (200 ± 82 versus 296 ± 65 mL · 100 g−1 · min−1, P < 0.05). Within a contusion-enriched ROI, CBF was reduced after TBI (142 ± 73 versus 280 ± 64 mL · 100 g−1 · min−1P < 0.05). Cerebral blood flow in the sham group was modestly reduced (212 ± 112 versus 262 ± 118 mL · 100 g−1 · min−1, P < 0.05). Also, TBI widened the distribution of CBF in injured and contralateral cortex. Hypocarbia reduced cortical CBF in control (48%), sham (45%), and TBI rats (48%) versus normocarbia, P < 0.05. In the contusion-enriched ROI, only controls showed a significant reduction in CBF, suggesting blunted CO2 reactivity in the sham and TBI group. CO2 reactivity was reduced in the sham (13%) and TBI (30%) groups within the cortical ROI (versus contralateral cortex). These values were increased twofold within the contusion-enriched ROI but were not statistically significant. After TBI, hypocarbia narrowed the CBF distribution in the injured cortex. We conclude that perfusion MRI using arterial spin-labeling is feasible for the serial, noninvasive measurement of CBF and CO2 reactivity in rats.

Early perfusion after controlled cortical impact in rats: quantification by arterial spin-labeled MRI and the influence of spin-lattice relaxation time heterogeneity.

Early posttraumatic cerebral hypoperfusion is implicated in the evolution of secondary damage after experimental and clinical traumatic brain injury (TBI). This is the first report of cerebral blood flow (CBF) measurement by continuous arterial spin‐labeled magnetic resonance imaging (MRI) early after TBI in rats using the controlled cortical impact (CCI) model. CCI reduced CBF globally at ∼3 hr (versus normal), with 85% and 49% reductions in a contused cortical region and contralateral cortex, respectively. In contrast, a prior MRI study from this laboratory showed at 24 hr post trauma a focal CBF reduction restricted to the injury site. In vivo spin‐lattice relaxation time (T1obs), which is used in CBF quantification, was spatially heterogeneous early after CCI, a time when edema is developing in injured brain tissue. At 4.7 T, T1obs values are increased 29% in the contusion (versus normal), consequently reducing CBF quantification to a similar degree. MRI should facilitate coupling posttraumatic CBF with long‐term functional outcome. Magn Reson Med 42:673–681, 1999.

Effects of Hypothermia on Traumatic Brain Injury in Immature Rats

Hypothermia is beneficial in adult models of traumatic brain injury (TBI), but it has not been evaluated in an immature animal model. We hypothesized that brief hypothermia applied after TBI would reduce cerebral edema and lesion volume in immature rats. Male Wistar rats (3–4 weeks of age, 90–140 g) were anesthetized, intubated, mechanically ventilated, and subjected to TBI by a weight drop onto the exposed right parietal cortex. Hypothermic rats were then cooled to a brain temperature of 32.0 ± 0.5°C for 4 h, and control rats were maintained at a brain temperature of 37.0 ± 0.5°C. Cerebral edema (wet — dry weight method) was assessed at 4 and 24 h, and lesion volume was assessed at 5 days. At 4 h, a reduction of percent brain water in the traumatized hemisphere was observed in hypothermic versus normothermic rats (81.75 ± 0.60 vs. 82.53 ± 0.67%; p < 0.05), but by 24 h posttrauma, the groups were similar (p = 0.82). Total lesion volume (47.2 ± 8.5 vs. 44.4 ± 10.0 mm3; p = 0.51) and necrotic volume (20.2 ± 6.3 vs. 20.0 ± 7.9 mm3; p = 0.95) were similar in the hypothermic and normothermic groups. We conclude that in this model, a transient (4-h) application of moderate (32°C) hypothermia reduces the cerebral edema characteristically seen in immature rats at 4 h, but this reduction is not sustained at 24 h. Attenuating or delaying the development of cerebral edema could have important therapeutic relevance after TBI. Transient hypothermia, however, did not reduce lesion volume at 5 days posttrauma.

Cerebral Blood Flow at One Year after Controlled Cortical Impact in Rats: Assessment by Magnetic Resonance Imaging

Progressive tissue loss and delayed cognitive deficits are seen in rats during the initial year after experimental traumatic brain injury (TBI). As much as 10% of parenchymal volume is lost even in the contralateral hemisphere by 1 year after controlled cortical impact (CCI) in rats. Progressive declines in cerebral blood flow (CBF) are also associated with advanced age and neurodegenerative diseases. Surprisingly, the long-term effects of TBI on CBF remain undefined. CBF was quantified by continuous arterial spin-labeled magnetic resonance imaging (MRI) and measurements of spin-lattice relaxation time in a slice through the plane of injury at 1 year after experimental TBI produced by CCI (n = 4) or sham surgery (n = 4) in rats. CBF was quantified in six regions of interest (ROIs) that were anatomically identified on the control images in each hemisphere and included a medial cortical segment (contusion-enriched, beneath the impact site, on the ipsilateral side) cortex, hippocampus, thalamus, amygdala/pyriform cortex, and hemisphere. At 1 year after injury, CBF was dramatically (96%) reduced in structures within the large cystic lesion that was seen in three of four rats and variably included cortex and hippocampus. Overall, there was an 80% reduction in CBF in the ipsilateral medial cortical segment comparing CCI and sham groups. Similarly, 52% and 67% reductions were seen in CBF in the cortical and hippocampal ROIs ipsilateral to impact (CCI vs. sham), respectively. These are regions both with marked CBF disturbances early after injury and that ultimately suffer considerable tissue loss over the 1-year interval. However, at 1 year after CCI, CBF was not different from sham in other ROIs, including ipsilateral thalamus, or either contralateral hippocampus or hemisphere. We conclude that, at 1 year after CCI, CBF is reduced in anatomic structures at or near the impact site, including injured cortex and hippocampus, and this translates into a reduction in hemispheric CBF. However, despite both significant occult tissue loss ipsilateral and contralateral to the injury and delayed cognitive deficits, widespread reductions in CBF are not observed. This suggests the possibility of remodeling or repackaging of the brain that preserves CBF outside of the cystic lesion.