Mengzhou Xue

Intracerebral injection of autologous whole blood in rats: time course of inflammation and cell death

Intracerebral hemorrhage is associated with stroke and head trauma. The purpose of this study was to study brain inflammation and cell death in adult rats 1 h to 4 weeks after injection of blood into the striatum. Terminal dUTP nick-end-labeling positive dying cells were evident 4 h to 4 weeks post-hemorrhage. Neutrophil infiltration was brief and peaked at 48 h. CD8a immunoreactive lymphocytes, possibly natural killer cells, became apparent at 48 h and persisted for 1 week. Microglial reaction was evident at 4 h and persisted for 4 weeks. We conclude that extravascular blood causes a mixed inflammatory cell reaction in brains that is maximal from 48-72 h following hemorrhage. This is associated with death of brain cells over a prolonged period of at least 4 weeks.

Antisense Oligodeoxynucleotide Inhibition of Tumor Necrosis Factor-α Expression Is Neuroprotective After Intracerebral Hemorrhage

Background and Purpose— Tumor necrosis factor-&agr; (TNF-&agr;) expression is increased in brain after cerebral ischemia, although little is known about its abundance and role in intracerebral hemorrhage (ICH). A TNF-&agr;–specific antisense oligodeoxynucleotide (ORF4-PE) was used to study the extent to which TNF-&agr; expression influenced neurobehavioral outcomes and brain damage in a collagenase-induced ICH model in rat. Methods— Male Sprague-Dawley rats were anesthetized, and ICH was induced by intrastriatal administration of heparin and collagenase. Immediately before or 3 hours after ICH induction, ORF4-PE was administered directly into the site of ICH. TNF-&agr; mRNA and protein levels were measured by reverse transcriptase–polymerase chain reaction and immunoblot analyses. Cell death was measured by terminal deoxynucleotidyl transferase–mediated uridine 5′triphosphate-biotin nick end labeling (TUNEL). Neurobehavioral deficits were measured for 4 weeks after ICH. Results— ICH induction (n=6) elevated TNF-&agr; mRNA and protein levels (P <0.01) at 24 hours after the onset of injury compared with sham controls (n=6). Immunohistochemical labeling indicated that ICH was accompanied by elevated expression of TNF-&agr; in neutrophils, macrophages, and microglia. Administration of ORF4-PE (2.0 nmol) directly into striatal parenchyma, 15 minutes before (n=4) or 3 hours after (n=6) ICH, decreased levels of TNF-&agr; mRNA (P <0.001) and protein (P <0.01) in the brain tissue surrounding the hematoma compared with animals treated with saline alone (n=6). Mean±SEM striatal cell death (cells per high-powered field) was also reduced in animals receiving ORF4-PE (34.1±5.0) compared with the saline-treated ICH group (80.3±7.50) (P <0.001). ORF4-PE treatment improved neurobehavioral deficits observed at 24 hours (P <0.001) after induction of ICH (n=6) compared with the untreated ICH group (n=6). This improvement was maintained at 28 days after hemorrhage induction (P <0.001). Conclusions— These results indicate a pathogenic role for TNF-&agr; during ICH and demonstrate that reducing TNF-&agr; expression using antisense oligodeoxynucleotides is neuroprotective.

Effect of FK-506 on Inflammation and Behavioral Outcome Following Intracerebral Hemorrhage in Rat

Beginning 15 min after induction of intracerebral hemorrhage (ICH) by intrastriatal administration of collagenase, rats were treated intramuscularly with FK-506 (3 mg/kg) or with vehicle. Treatment was repeated daily for 7 days. MR imaging 1, 7, and 28 days post-ICH showed that treatment did not affect hematoma size or its subsequent resolution. Two days post-ICH, neutrophil infiltration around the hematoma was decreased in the FK-506-treated rats, as was the number of TUNEL-positive cells at the edge of the hematoma and in the peripheral region. The decreased inflammatory response was accompanied by functional improvement in the treated rats. The neurological deficit induced by the ICH (beam walking ability, postural reflex, spontaneous circling) was significantly decreased from 3 to 21 days post-ICH by treatment with FK-506. Skilled use of the forelimb ipsilateral to the ICH was improved and sensory neglect of the same limb was decreased 8-9 weeks post-ICH in rats treated with FK-506. However, neuronal loss assessed 9 weeks post-ICH was not different in the treated and untreated rats.

Comparison of brain cell death and inflammatory reaction in three models of intracerebral hemorrhage in adult rats

Intracerebral hemorrhage (ICH) is associated with stroke and head trauma. Different experimental models are used, but it is unclear to what extent the tissue responses are comparable. The purpose of this study was to compare the temporal responses to brain hemorrhages created by injection of autologous whole blood, collagenase digestion of blood vessels, and avulsion of cerebral blood vessels. Adult rats were subjected to ICH. Rats were perfusion fixed with paraformaldehyde 1 hour to 28 days later. Hematoxylin and eosin, Fluoro-Jade, immunohistochemical, and TUNEL staining were used to allow quantification of damaged and dying neurons, neutrophils, CD8alpha immunoreactive lymphocytes, and RCA-1 positive microglia/macrophages, adjacent to the hemorrhagic lesion. In all models, eosinophilic neurons peaked between 2 and 3 days. TUNEL positive cells were observed maximal at 2 days in blood injection model, 3 days in vessel avulsion model, between 1 and 7 days in the collagenase injection model, and were evident in small quantities in 21 to 28 days in 3 models. Neutrophils appeared briefly from 1 to 3 days in all models, but they were substantially lower in the cortical vessel avulsion model, perhaps owing to the devitalized nature of the tissue. Influx of CD8alpha immunoreactive lymphocytes were maximal at 2 to 3 days in the autologous injection model, 3 to 7 days in other 2 models, and persisted for 21 to 28 days in all models. The microglial/macrophage reaction peaked between 2 and 3 days in the blood injection model and at 3 to 7 days in other 2 models, and persisted for weeks in all groups. These results suggest that different models of ICH are associated with similar temporal patterns of cell death and inflammation. However, the relative magnitude of these changes differs.

Injections of Blood, Thrombin, and Plasminogen More Severely Damage Neonatal Mouse Brain Than Mature Mouse Brain

The mechanism of brain cell injury associated with intracerebral hemorrhage may be in part related to proteolytic enzymes in blood, some of which are also functional in the developing brain. We hypothesized that there would be an age‐dependent brain response following intracerebral injection of blood, thrombin, and plasminogen. Mice at 3 ages (neonatal, 10‐day‐old, and young adult) received autologous blood (15, 25, and 50 μl respectively), thrombin (3, 5, and 10 units respectively), plasminogen (0.03, 0.05, and 0.1 units respectively) (the doses expected in same volume blood), or saline injection into lateral striatum. Forty‐eight hours later they were perfusion fixed. Hematoxylin and eosin, lectin histochemistry, Fluoro‐Jade, and TUNEL staining were used to quantify changes related to the hemorrhagic lesion. Damage volume, dying neurons, neutrophils, and microglial reaction were significantly greater following injections of blood, plasminogen, and thrombin compared to saline in all three ages of mice. Plasminogen and thrombin associated brain damage was greatest in neonatal mice and, in that group unlike the other 2, greater than the damage caused by whole blood. These results suggest that the neonatal brain is relatively more sensitive to proteolytic plasma enzymes than the mature brain.

Persistent motor deficit following infusion of autologous blood into the periventricular region of neonatal rats

Periventricular hemorrhage (PVH) in the brain of premature infants is often associated with developmental delay and persistent motor deficits. Our goal is to develop a rodent model that mimics the behavioral phenotype. We hypothesized that autologous blood infusion into the periventricular germinal matrix region of neonatal rats would lead to immediate and long-term behavioral changes. Tail blood or saline was infused into the periventricular region of 1-day-old rats. Magnetic resonance (MR) imaging was used to demonstrate the hematoma. Rats with blood infusion, as well as saline and intact controls, underwent behavior tests until 10 weeks age. Blood-infused rats displayed significant delay in motor development (ambulation, righting response, and negative geotaxis) to 22 days of age. As young adults, they exhibited impaired ability to stay on a rotating rod and to reach for food pellets. MR imaging at 10 weeks demonstrated subsets of rats with normal appearing brains, focal cortical infarcts, or mild hydrocephalus. There was a good correlation between MR imaging and histological findings. Some rats exhibited periventricular heterotopia and/or subtle striatal abnormalities not apparent on MR images. We conclude that autologous blood infusion into the brain of neonatal rats successfully models some aspects of periventricular hemorrhage that occurs after premature birth in humans.

Does Thrombin Play a Role in the Pathogenesis of Brain Damage After Periventricular Hemorrhage

Neonatal periventricular hemorrhage (PVH) is a devastating complication of prematurity in the human infant. Based upon observations made primarily in adult rodents and the fact that the immature brain uses proteolytic systems for cell migration and growth, we hypothesized that thrombin and plasmin enzyme activities contribute to the brain damage after PVH. The viability of mixed brain cells derived from newborn rat periventricular region was suppressed by whole blood and thrombin, but not plasmin. Following injection of autologous blood into the periventricular region of newborn rat brain, proteolytic activity was detected in a halo around the hematoma using membrane overlays impregnated with thrombin and plasmin fluorogenic substrates. Two‐day old rats received periventricular injection of blood, thrombin, and plasminogen. After 2 days, thrombin and blood were associated with significantly greater damage than saline or plasminogen. Two‐day old mice received intracerebral injections of blood in combination with saline or the proteolytic inhibitors hirudin, α2macroglobulin, or plasminogen activator inhibitor‐1. After 2 days, hirudin significantly reduced brain cell death and inflammation. Two‐day‐old mice then received low and high doses of hirudin mixed with blood after which behavioral testing was conducted repeatedly. At 10 weeks there was no statistically significant evidence for behavioral or structural brain protection. These results indicate that thrombin likely plays a role in neonatal periventricular brain damage following PVH. However, additional factors are likely important in the recovery from this result.

Intracerebral infusion of a second-generation ciliary neurotrophic factor reduces neuronal loss in rat striatum following experimental intracerebral hemorrhage

Neuronal and glial cell death in the striatum of a rat model of collagenase-induced intracerebral hemorrhage begins at 1 day and continues for at least 3 weeks. We hypothesized that administration of a neurotrophic agent would reduce neuronal loss in this experimental model. Because it has been shown to protect striatal neurons against excitotoxic injury, a second-generation ciliary neurotrophic factor (CNTF) (AXOKINE) was administered by continuous intracerebral infusion (2 microg/day) beginning 28 h after hemorrhage and continuing for 2 weeks. Magnetic resonance imaging showed that the hematoma size was comparable in control and treated rats prior to treatment. Counts of medium-sized striatal neurons within 320 microm of the hematoma 8 weeks after the hemorrhage revealed a slight but statistically significant benefit with a 42.5% loss in treated rats compared to 51.7% loss in controls. The results suggest that AXOKINE might be protective of striatal neurons in the vicinity of a hemorrhagic lesion.