Ofelia F. Alonso

Middle Cerebral Artery Occlusion in the Rat by Intraluminal Suture: Neurological and Pathological Evaluation of an Improved Model

BACKGROUND AND PURPOSE The purpose of the present study was to evaluate a modified method of intraluminal suture occlusion of the middle cerebral artery (MCA) on the volume of brain infarction and on neurobehavioral function in rats subjected to a temporary focal ischemic insult. METHODS Male Sprague-Dawley rats were anesthetized with halothane and subjected to 60 minutes or 2 hours of temporary MCA occlusion (MCAo) by an intraluminal thread. In one group of rats, the suture was coated with poly-L-lysine, while in a second group, a conventional uncoated suture was used. Behavioral function was evaluated at 50 to 60 minutes after occlusion and during a 3-day period after MCAo. Three days after MCAo brains were perfusion-fixed and infarct volumes were measured. RESULTS In rats with 60-minute MCAo, only 3 of 7 animals with uncoated sutures had infarcts, whereas in the group with poly-L-lysine-coated sutures, all rats (n = 7) exhibited infarction (P = .009, Fishers exact test). With 2 hours of MCAo, total infarct volume (corrected for brain edema) was significantly larger in rats with poly-L-lysine-coated sutures than in the group with uncoated sutures (mean +/- SEM, 122.1 +/- 4.8 versus 67.0 +/- 18.2 mm3, respectively; P = .03; n = 4 in each group). In the 2-hour MCAo study, infarct volumes in the uncoated-suture group tended to be variable and inconsistent (coefficient of variation, 54%) compared with the group in which sutures were coated with poly-L-lysine, in which a highly consistent infarct was produced (coefficient of variation of infarct volume, 8%). CONCLUSIONS Reversible MCAo in which a poly-L-lysine-coated intraluminal suture was used proved to be a reliable and effective modification of this technique, yielding consistently larger infarcts and greatly reduced interanimal variability.

Glutamate Release and Free Radical Production Following Brain Injury: Effects of Posttraumatic Hypothermia

Abstract: Posttraumatic hypothermia reduces the extent of neuronal damage in remote cortical and subcortical structures following traumatic brain injury (TBI). We evaluated whether excessive extracellular release of glutamate and generation of hydroxyl radicals are associated with remote traumatic injury, and whether posttraumatic hypothermia modulates these processes. Lateral fluid percussion was used to induce TBI in rats. The salicylate‐trapping method was used in conjunction with microdialysis and HPLC to detect hydroxyl radicals by measurement of the stable adducts 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA). Extracellular glutamate was measured from the same samples. Following trauma, brain temperature was maintained for 3 h at either 37 or 30°C. Sham‐trauma animals were treated in an identical manner. In the normothermic group, TBI induced significant elevations in 2,3‐DHBA (3.3‐fold, p < 0.01), 2,5‐DHBA (2.5‐fold, p < 0.01), and glutamate (2.8‐fold, p < 0.01) compared with controls. The levels of 2,3‐DHBA and glutamate remained high for approximately 1 h after trauma, whereas levels of 2,5‐DHBA remained high for the entire sampling period (4 h). Linear regression analysis revealed a significant positive correlation between integrated 2,3‐DHBA and glutamate concentrations (p < 0.05). Posttraumatic hypothermia resulted in suppression of both 2,3‐ and 2,5‐DHBA elevations and glutamate release. The present data indicate that TBI is followed by prompt increases in both glutamate release and hydroxyl radical production from cortical regions adjacent to the impact site. The magnitude of glutamate release is correlated with the extent of the hydroxyl radical adduct, raising the possibility that the two responses are associated. Posttraumatic hypothermia blunts both responses, suggesting a mechanism by which hypothermia confers protection following TBI.

Intraischemic but Not Postischemic Brain Hypothermia Protects Chronically following Global Forebrain Ischemia in Rats

We investigated whether postischemic brain hypothermia (30°C) would permanently protect the hippocampus following global forebrain ischemia. Global ischemia was produced in anesthetized rats by bilateral carotid artery occlusion plus hypotension (50 mm Hg). In the postischemic hypothermic group, brain temperature was maintained at 37°C during the 10-min ischemic insult but reduced to 30°C starting 3 min into the recirculation period and maintained at 30°C for 3 h. In normothermic animals, intra- and postischemic brain temperature was maintained at 37°C. After recovery for 3 days, 7 days, or 2 months, the extent of CA1 hippocampal histologic injury was quantitated. At 3 days after ischemia, postischemic hypothermia significantly protected the hippocampal CA1 sector compared with normothermic animals. For example, within the medial, middle, and lateral CA1 subsectors, the numbers of normal neurons were increased 20-, 13-, and 9-fold by postischemic hypothermia (p < 0.01). At 7 days after the ischemic insult, however, the degree of postischemic hypothermic protection was significantly reduced. In this case, the numbers of normal neurons were increased an average of only threefold compared with normothermia. Ultrastructural analysis of 7-day postischemic hypothermic rats demonstrated CA1 pyramidal neurons showing variable degrees of injury surrounded by reactive astrocytes and microglial cells. At 2 months after the ischemic insult, no trend for protection was demonstrated. In contrast to postischemic hypothermia, significant protection was seen at 2 months following intraischemic hypothermia. These data indicate that intraischemic, but not postischemic, brain hypothermia provides chronic protection to the hippocampus after transient brain ischemia. The inability of postischemic hypothermia to protect chronically after 3 days could indicate that (a) postischemic hypothermia merely delays ischemic cell death and/or (b) the postischemic brain undergoes a secondary insult. In postischemic treatment protocols, chronic survival studies are required to determine accurately the ultimate histopathological outcome following global cerebral ischemia.

Post-traumatic brain hypothermia reduces histopathological damage following concussive brain injury in the rat

The purposes of this study were (1) to document the histopathological consequences of moderate traumatic brain injury (TBI) in anesthetized Sprague-Dawley rats, and (2) to determine whether posttraumatic brain hypothermia (30°C) would protect histopathologically. Twenty-four hours prior to TBI, the fluid percussion interface was positioned over the right cerebral cortex. On the 2nd day, fasted rats were anesthetized with 70% nitrous oxide, 1% halothane, and 30% oxygen. Under controlled physiological conditions and normothermic brain temperature (37.5°C), rats were injured with a fluid percussion pulse ranging from 1.7 to 2.2 atmospheres. In one group, brain temperature was maintained at normothermic levels for 3 h after injury. In a second group, brain temperature was reduced to 30°C at 5 min post-trauma and maintained for 3 h. Three days after TBI, brains were perfusion-fixed for routine histopathological analysis. In the normothermic group, damage at the site of impact was seen in only one of nine rats. In contrast, all normothermic animals displayed necrotic neurons within ipsilateral cortical regions lateral and remote from the impact site. Intracerebral hemorrhagic contusions were present in all rats at the gray-white interface underlying the injured cortical areas. Selective neuronal necrosis was also present within the CA3 and CA4 hippocampal subsectors and thalamus. Post-traumatic brain hypothermia significantly reduced the overall sum of necrotic cortical neurons (519±122 vs 952±130, mean ±SE, P=0.03, Kruskal-Wallis test) as well as contusion volume (0.50±0.14 vs 2.14±0.71 mm3, P=0.004). These data document a consistent pattern of histopathological vulnerability following normothermic TBI and demonstrate hypothermic protection in the post-traumatic setting.

Moderate hyperglycemia worsens acute blood-brain barrier injury after forebrain ischemia in rats.

Background and Purpose Clinical and experimental data indicate that hyperglycemia can aggravate the consequences of stroke and cerebral ischemia. The purpose of this study was to examine the effects of moderate hyperglycemia on the response of the blood-brain barrier to normothermic (37°C) and hypothermic (30°C) global forebrain ischemia. Methods Sixteen rats underwent 20 minutes of four-vessel occlusion followed by 30 minutes of postischemic recirculation. We used the protein tracer horseradish peroxidase as an indicator of increased vascular permeability, and rats were perfusion-fixed for microscopic analysis. To produce moderate hyperglycemia, we gave an intraperitoneal injection of 50% dextrose 15 minutes before the ischemic insult. Results After normothermic brain ischemia, normoglycemico rats (plasma glucose level, 115 ± 3 mg/dl) demonstrated extravasated horseradish peroxidase mainly restricted to the cerebral cortex. In contrast, more severe and widespread protein extravasation was documented throughout the neuraxis of hyperglycemic (plasma glucose level, 342 ± 27) rats. Sites of protein leakage included the cerebral cortex, striatum, hippocampus, thalamus, and cerebellum. Foci of protein extravasation were associated with pial and large penetrating vessels. Intraischemic hypothermia significantly attenuated the blood-brain barrier consequences of hyperglycemic brain ischemia. Conclusions Under normothermic ischemic conditions, hyperglycemia significantly worsens the degree of acute blood-brain barrier breakdown compared with normoglycemia. Postischemic blood-brain barrier disruption may play an important role in the pathogenesis of increased brain damage associated with systemic hyperglycemia.

Delayed posttraumatic brain hyperthermia worsens outcome after fluid percussion brain injury: a light and electron microscopic study in rats.

The morphological consequences of delayed posttraumatic brain hyperthermia (39 degrees C) after fluid percussion brain injury were assessed in rats. Sprague-Dawley rats anesthetized with 4% halothane and maintained on a 70:30 mixture of nitrous oxide:oxygen and 0.5% halothane underwent moderate (1.5-2.0 atm) traumatic brain injury with the injury screw positioned parasagittally over the right parieto-occipital cortex. At 24 hours after traumatic brain injury, the rats were reanesthetized and randomized into two groups in which either a 3-hour period of brain normothermia (36.5 degrees C, n = 18) or hyperthermia (39 degrees C, n = 18) was maintained. Sham-operated controls (n = 10) underwent all surgical and temperature-monitoring procedures. After the 3-hour monitoring period, the rats were allowed to survive for 3 days for light microscopic analysis or were injected with the protein tracer horseradish peroxidase and were perfusion-fixed 15 minutes later for light and electron microscopic analysis. At 4 days after traumatic brain injury, delayed posttraumatic hyperthermia (n = 12) significantly increased mortality (47%) and contusion volume (1.7 +/- 0.69 mm3, mean +/- standard error of the mean), compared to normothermia (n = 12) (18% mortality and 0.13 +/- 0.21 mm3 contusion volume) (P < 0.01, analysis of variance). At 15 minutes after the 3-hour hyperthermic period, the area of hemorrhage and horseradish peroxidase extravasation overlying the lateral external capsule was significantly increased (2.52 +/- 0.71 mm2, mean +/- standard error of the mean, versus 0.43 +/- 0.16 mm2) (P < 0.01), compared to normothermic rats. Examination of toluidine blue-stained plastic sections demonstrated a higher frequency of abnormally swollen myelinated axons per high microscopic field with hyperthermia. For example, numbers of swollen axons within the sixth layer of the right somatosensory cortex, corpus callosum, and internal capsule were 7.3 +/- 1.3, 4.2 +/- 1.4, and 3.0 +/- 1.2 axons (mean +/- standard error of the mean) with normothermia, respectively, compared with 24.7 +/- 12.1, 33.1 +/- 4.2, and 27.3 +/- 3.1 axons with hyperthermia, respectively (P < 0.01). An ultrastructural examination of the swollen axons demonstrated a severely thinned myelin sheath containing axoplasm devoid of cytoskeletal components. These experimental results indicate that posttraumatic brain hyperthermia might increase morbidity and mortality in patients with head injury by aggravating axonal and microvascular damage.

Importance of posttraumatic hypothermia and hyperthermia on the inflammatory response after fluid percussion brain injury : Biochemical and immunocytochemical studies

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 ± 56.2 versus control 65.5 ± 52.3 U/g of wet tissue; mean ± SD) and posterior (injured) cortical and subcortical segments compared to shamoperated rats (305.76 ± 27.8 and 258.67 ± 101.4 U/g of wet tissue versus control 62.8 ± 24.8 and 37.28 ± 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 ± 258.4 and 100.11 ± 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.

Therapeutic Neutralization of the NLRP1 Inflammasome Reduces the Innate Immune Response and Improves Histopathology after Traumatic Brain Injury

Traumatic brain injury elicits acute inflammation that in turn exacerbates primary brain damage. A crucial part of innate immunity in the immune privileged central nervous system involves production of proinflammatory cytokines mediated by inflammasome signaling. Here, we show that the nucleotide-binding, leucine-rich repeat pyrin domain containing protein 1 (NLRP1) inflammasome consisting of NLRP1, caspase-1, caspase-11, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), the X-linked inhibitor of apoptosis protein, and pannexin 1 is expressed in neurons of the cerebral cortex. Moderate parasagittal fluid-percussion injury (FPI) induced processing of interleukin-1β, activation of caspase-1, cleavage of X-linked inhibitor of apoptosis protein, and promoted assembly of the NLRP1 inflammasome complex. Anti-ASC neutralizing antibodies administered immediately after fluid-percussion injury to injured rats reduced caspase-1 activation, X-linked inhibitor of apoptosis protein cleavage, and processing of interleukin-1β, resulting in a significant decrease in contusion volume. These studies show that the NLRP1 inflammasome constitutes an important component of the innate central nervous system inflammatory response after traumatic brain injury and may be a novel therapeutic target for reducing the damaging effects of posttraumatic brain inflammation.

Apoptotic and Antiapoptotic Mechanisms After Traumatic Brain Injury

Caspase and inhibitor of apoptosis (IAP) expression was examined in rats subjected to moderate traumatic brain injury (TBI) using a parasagittal fluid-percussion brain insult (1.7 to 2.2 atm). Within 1 hour after injury, caspase-8 and −9, two initiators of apoptosis, were predominantly expressed in superficial cortical areas adjacent to the impact site and in the thalamus. Caspase-3, an effector caspase, was evident at 6 hours throughout the traumatized cerebral cortex and hippocampus. Moreover, the authors observed that XIAP, cIAP-1, and cIAP-2, members of the IAP family, were constitutively expressed in the brain. Colocalization of XIAP-immunolabled cells with cell-specific markers indicated that XIAP is expressed within neurons and a subpopulation of oligodendrocytes. Immunoblots of brain extracts revealed that the processed forms of caspase-8, −9, and −3 are present as early as 1 hour after trauma. The appearance of activated caspases corresponded with the detection of cleavage of XIAP into fragments after injury and a concomitant increase in the levels of cIAP-1 and cIAP-2 in the traumatized hemispheres. The current data are consistent with the hypotheses that caspases in both the extrinsic and intrinsic apoptotic pathways are activated after moderate TBI and that IAPs may have a protective role within the brain with alterations in levels and cleavage of IAPs that contribute to cell death in this setting.

A pictorial representation of endogenous brain ATP by a bioluminescent method

The layering of a luciferin-luciferase solution on brain slices makes endogenous ATP visible. Rat brains, frozen in situ and sliced at 16 micrometer thickness at a temperature of--15 degrees C, were fixed by a ternary mixture of ethanol, formalin and dioxane at--20 degrees C for 15 min, and dried at 40 degrees C for 12 h. Luciferin, luciferase and a small quantity of magnesium sulfate were dissolved into an arsenate buffer solution containing 1% polyvinylpyrrolidone, 2% gelatin and 1% glycerol. The solution was then frozen into a column, sliced at 40 micrometer thickness at--15 degrees C and placed on the precooled brain slice. A luminiferous luciferin-ATP reaction begins when the brain slice reaches room temperature and persists for more than 10 min. This technique therefore makes possible the optical and/or photographic determination of the endogenous concentration of brain ATP. Capability of the technique is also demonstrated.