The Experimental Study On The Perihematomal Penumbra And Its Time Window

Cerebroascular diseases are the most common diseases imperiling human being with high rate of incidence, mortality and deformity. Recently, lots of achievements were made in experimental and clinical researches on the cerebroascular diseases, as was instanced by the discovery of ischemic penumbra, which laid a foundation of thrombolytic therapy. In contrast to the ischemic stroke, victims of intracerebral hemorrhage (ICH) commonly adopted canonical and conservative treatments, suffering higher mortality and more severe deficits. The poor outcome is mainly ascribed to increased intracranial pressure and secondary neuronal injury in the perihematomal tissue. Space-occupying effect, neurotoxicity, and ischemic damnification might be involved in the secondary neuronal injury. Recently, perihematomal penumbra has turn into one of the focuses of ICH research for its importance in the strategy of ICH management, which carrying on as one of the most arduous and pivotal work in the ICH research. If the perihematomal penumbra and its time window were confirmed, the strategy of ICH management be modulated thoroughly.The concept of penumbra was brought forward by Astrup in 1977. Its main characteristics are ischemia, reversibility and time widow. The penumbra tissue suffersreversible ischemic damage, which would turn into irreversible sclarosis in a definite time limit.The results of perihematomal penumbra studies are arguing. Some zoopery and clinical imaging investigations suggested the existence of an ischemic penumbra surrounding the hematoma. On the contrast, other experiments failed to demonstrate consistent ischemia areas in the peripheral to the hematoma. The blood flow was not found beyond the threshold for the ischemic injury (15-25 ml/lOOg/min), or beyond the threshold only for a short time. The blood flow returned to normal or even higher level within 3-4 hours. These results suggested as if critical levels and durations of hypoperfusion do not occur after experimental ICH. As we know, the most important pathological effects of hematoma are from the mass effect and the toxic substances of clot. We found most of these experiments introduced the animal model injected with fresh autologous blood. Because of the blood countercurrent along the needle track, they shared the problems of the lack of mass effect and poor reproducibility of hematoma, which could mislead us into the different conclusion. Contrariwise, significant reduction in the cerebral blood flow (below 20 or 25 ml/lOOg/min) was revealed in the ipsilateral caudate nucleus of ICH animal model induced by mechanical microballoon to simulate intracerebral hemorrhage. Accordingly, animal model would be the key of the experiments of perihematomal penumbra. In the current research, we modified the ICH animal model by injecting clotted autologous blood, compared it with the double-injection model, demonstrated perihematomal penumbra and evaluated its time window, evaluated the effects of hematoma aspiration on the perihematomal penumbra, revealed the malfunction of microcirculation in the perihematomal penumbra.1. An intracerebral hemorrhage model in rats by injecting clotted autologous blood into caudate nucleusTwenty adult male SD rats were arranged into 4 groups: Eight in eachexperimental group (clotted, double-injection), two in each control group (Sham, target-proof). The matching design was adopted in the study. Paired rats were from the same brood with similar weight. 50 u.L clotted autologous blood was injected into the stereotactic target in the middle of the right caudate nucleus (3 mm lateral, 1 mm anterior, 6 mm ventral to the bregma) of the rats in the clotted group. Rats in the double-injection group underwent the same stereotactic operation procedure. The injection procedure of blood was different. 50 \xL fresh blood was injected slowly into the right caudate nucleus in two separate procedures. Each of the rats was re-anesthetized and killed by cardiac perfusion with 4% paraformaldehyde 6 hours after operation. After perfusion fixation, brain was removed and immersed into 10% formalin over night for further fixation. Coronal slices of 1 mm thick were cut with a matrix. The images of serial cerebral slices were input into image analysis system to count the volume of hematoma.Each rat of the clotted group showed a hematoma of similar size and shape in striatum with distinct mass effect. The hematoma took on an ovoid shape occupying approximately half of the cross-sectional area of the striatum, mildly extending into callose. The primary hematoma in the striatum of the double-injection group was relatively small, with severe blood overflow into callose, ventricle and subarachnoid space. The hematoma volume of the clotted group was larger than that of the double-injection group. The difference between the primary hematoma volume of the two groups was significant (P<0.001), so was the overall hematoma volume (P<0.05). That meant there's more blood leaking out into subarachnoid space in the double-injection group.The components of blood coagulated outside body are the same as inside, so that the biochemical effects wouldn't be impaired. The hematoma of the clotted group was in possession of distinct mass effect and better reproducibility than that of the double-injection group, which is more similar with human ICH. It would satisfy the need of experimental ICH studies of every field, especially of perihematomal penumbra.2. The study on the perihematomal penumbra: Distribution and its time windowForty-eight adult male SD rats were arranged into 4 experimental groups (2-hour, 3-hour, 4-hour and 6-hour), and two in the control group. The matching design was adopted in the study. Clotted autologous blood rat model of ICH was adopted. The operation procedure was the same as part one. Similar procedure was taken to the control group except the injection of blood. Rats were re-anesthetized after the corresponding time with the number of the group (2 hours, 3 hours, 4 hours, 6 hours), and killed by decapitation. Coronal brain slices of 2 mm thick were cut with a matrix. The two pieces adjacent to the needle entry site were reserved for the study of perihematomal penumbra evaluation. The forward slice was stained by 2% triphenyltetrazolium chloride (TTC) in PBS for 15 minutes (37 V), and subsequently fixed in 10% formalin in PBS. The backward slice employed for examination of cell morphology was quickly frozen with a cool stage. Sections were cut at the needle entry end with a cryomicrotome and stained with hematoxylin and eosin (HE). The lesions were measured with the Mias image analysis system. The following areas were identified: area of hematoma, overall area of ischemic injury, area of necrosis. The overall area of ischemic injury was identified with the images of TTC staining, and the area of necrosis with the images of HE staining. The area of penumbra was that the ischemic area minus the necrotic. The area ratio of penumbra to ischemia was output as penumbra index for statistics.The tissue around the hematoma looked pale and areolar after HE staining. There's only slight ischemic damnification found in the 2-hour group, including swelling, nuclear asymmetry and pyknosis mainly in neurons peripheral to the hematoma. Neuronal necrosis progressed remarkably after 3 hours, spreading out as far as the most of the ischemic territory. Neurons and astrocytes are fragmented, accompanied by neuronophagia, satellite phenomenon, glia proliferation and neutrophile infiltration. Cell morphology was normal in slices of the sham control.The penumbra area in the 2-hour group was covered almost the overall ischemic territory, but contracted enormously in the 3-hour group, few and far between in the 4-hour and 6-hour groups. It could be assessed primarily that the time window of the perihematomal penumbra be about 4 hours. Quantitative analysis indicateded that the penumbra index of the 6-hour group was significantly lower than that of the 2-hour and 3-hour groups except the 4-hour group, which confirmed that the time window of perihematomal penumbra be about 4 hours.The existence of perihematomal penumbra has been confirmed. Its time window is about 4 hours, in the similitude of the cerebral infarction penumbra. The existence of the perihematomal penumbra and its time window remind us the necessity of ultra-early evacuation of hematoma.3. Intervening on the perihematomal penumbra: Effects of ultra-early hematoma aspirationTwenty-four adult male SD rats were arranged into 2 experimental groups (1-hour, 4-hour). The matching design was adopted in the study. Clotted autologous blood rat model of ICH was adopted. The operation procedure was the same as part one. The hematoma was aspired after lysis with Urokinase afer correspond time (1 hour, 4 hours). Area of ischemic tissue was shown with TTC staining. The lesions were measured with the Mias image analysis system.It's shown by either TTC and HE staining that the area of ischamic damaged peihematomal tissue in 1-hour group was significantly less than that in 4-hour group.The ultra-early aspiration of hematoma is effective to save the perihematomal penumbra, while the important character of reversibility is proved.4. The pathophysiological study on the perihematomal penumbraThirty-six adult male SD rats were arranged into 3 groups (hematoma, aspiration,sham). The matching design was adopted in the study. Clotted autologous blood rat model of ICH was adopted. The rats of aspiration group underwent hematomal aspiration 1 hour after operation. Each of the rats was re-anesthetized and killed by cardiac perfusion with 4% paraformaldehyde (under pressure of 90 mmHg) 6 hours after operation. After perfusion fixation, they were perfused with indian-ink/dextran mixture under the same pressure, and subsequently fixed in 10% formalin/PBS. Sections of 100 jim thick were cut with a cryomicrotome and examed directly after dehydrate and clarity. The mean dimension of capillary vessel and index of capillary bed (the area ratio of capillary bed in each field) in the perihematomal tissue were measured with the Mias image analysis system.The capillary bed decreased in the perihematomal tissue in the hematoma group, while capillary vessel narrowed, silt and looked stiff. Lots of capillary vessels split with ink leakage. The mean dimension of capillary vessel in the perihematomal tissue in hematoma group was only 52.8% of that in the sham group, while the same between the two groups of hematoma and aspiration. The index of capillary bed in the perihematomal tissue of hematoma group was significantly reduced compared with the sham and aspiration group.Obviously, there's impaired microcirculation in the perihematomal tissue, which capillary vessels narrowed, silt and looked stiff. The capillary bed in the perihematomal tissue was significantly reduced. The ultra-early aspiration of hematoma could improve the microcirculation of perihematomal penumbra, while reperfusion damage probably occurs in the perihematomal penumbra.