Microvascular Clip Research Articles

Behavioral characterization and effect of clinical drugs in a rat model of pain following spinal cord compression

Chronic pain symptoms, including spontaneous unevoked pain and evoked cutaneous hypersensitivity, appear following spinal cord injury (SCI). A reliable preclinical model is needed to develop effective analgesic treatments for these symptoms. A previously described rat model of SCI pain was modified and behaviorally characterized and used to test clinically available drugs. A segment of the mid-thoracic spinal cord was compressed for 60 s with a micro-vascular clip. The sensitivity of the hind paws to noxious heat (Hargreaves test), innocuous tactile (von Frey filaments), and cooling (acetone) stimuli were determined once per week beginning 1 week following spinal compression. Spinal cord compression led to long lasting hypersensitivity to stimuli, lasting for at least 12 weeks post-surgery. Systemic baclofen, gabapentin, tramadol, and morphine dose-dependently attenuated tactile hypersensitivity. No effect on tactile hypersensitivity was observed with amitriptyline, carbamazepine, rofecoxib, and diazepam. Baclofen and morphine also dose-dependently ameliorated heat hypersensitivity. In contrast, no effect on heat hypersensitivity was observed with amitriptyline, carbamazepine, diazepam and gabapentin. The current data suggest that the model can potentially differentiate those drugs with analgesic efficacy from those that do not have efficacy in SCI pain. Thus, the model may be useful for rapid screening and clinical translation of promising SCI pain therapies.

Adrenomedullin and Adrenomedullin Binding Protein-1 Prevent Acute Lung Injury after Gut Ischemia-Reperfusion

Ischemic bowel remains a critical problem, resulting in up to 80% mortality. Acute lung injury, a common complication after intestinal ischemia/reperfusion (I/R), might be responsible for such a high mortality rate. Our previous studies have shown that administration of a novel vasoactive peptide adrenomedullin (AM) and its binding protein (AMBP-1) reduces the systemic inflammatory response in rat models of both hemorrhage and sepsis. It remains unknown whether administration of AM/AMBP-1 has any protective effects on intestinal I/R-induced acute lung injury. We hypothesized that administration of AM/AMBP-1 after intestinal I/R prevents acute lung injury through downregulation of proinflammatory cytokines. Intestinal I/R was induced by placing a microvascular clip across superior mesenteric artery (SMA) for 90 minutes in adult male Sprague-Dawley rats (275 to 325 g). On release of the SMA clamp, the animals were treated with either AM (12 mug/kg body weight) in combination with AMBP-1 (40 microg/kg body weight) or vehicle (1 mL normal saline) during a period of 30 minutes through a femoral vein catheter. Lung samples were collected at 4 hours after treatment or sham operation. Lung injury was assessed by examining lung water content, morphologic changes, and granulocyte myeloperoxidase activity. Tumor necrosis factor-alpha and interleukin-6 gene expression and their protein levels in the lungs were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In additional groups of animals, AM/AMBP-1 or vehicle was administered at 1 hour after onset of reperfusion. Lung histology was examined at 3 hours after treatment. Intestinal I/R induced considerable lung injury, as characterized by lung edema, histopathologic changes, increased myeloperoxidase activity, and proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) levels in the lungs. Administration of AM/AMBP-1 after ischemia mitigated lung injury and dramatically downregulated proinflammatory cytokines. Lung injury was also ameliorated by delayed AM/AMBP-1 treatment as evidenced by improvement in lung histology. AM/AMBP-1 can be developed as a novel treatment to attenuate acute lung injury after an episode of gut ischemia. The protective effect of AM/AMBP-1 appears to be mediated through downregulation of proinflammatory cytokines.

Adrenomedullin and adrenomedullin-binding protein-1 downregulate inflammatory cytokines and attenuate tissue injury after gut ischemia-reperfusion

Recent studies have shown that adrenomedullin (AM) and AM-binding protein-1 (AMBP-1) possess anti-inflammatory properties in sepsis. We hypothesized that administration of AM/AMBP-1 after gut ischemia-reperfusion (I/R) downregulates inflammatory cytokines and attenuates tissue injury. Male Sprague-Dawley rats (275-325 g) were used. Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery (SMA) for 90 minutes. Upon release of the SMA clamp, the animals were treated by AM (12 microg per kilogram of body weight) and AMBP-1 (40 microg per kilogram of body weight) in combination, or vehicle (1 mL 0.9% NaCl) over 30 minutes via a femoral vein catheter. The animals undergoing sham operation or ischemia for 90 minutes only, did not receive AM/AMBP-1 treatment. At 60 minutes after the completion of the treatment (ie, 90 minutes after reperfusion), blood samples were collected. Plasma AM and AMBP-1 were measured by radioimmunoassay and Western blot analysis, respectively. Serum levels of TNF-alpha, interleukin (IL)-1beta, IL-6, IL-10, transaminases (ie, alanine aminotransaminase, aspartate aminotransaminase), lactate, and creatinine were determined with the use of enzyme-linked immunosorbent assay and other standard methods. In additional groups of animals, the 10-day survival rate was recorded after gut I/R. Ischemia alone was sufficient to downregulate both AM and AMBP-1. Unlike AMBP-1 that remained decreased, AM levels increased significantly after reperfusion. I/R but not ischemia alone significantly increased serum levels of inflammatory cytokines. Moreover, I/R-induced tissue injury was evidenced by increased levels of transaminases, lactate, and creatinine. Administration of AM/AMBP-1 after ischemia, however, markedly reduced cytokine levels, attenuated tissue injury, and improved survival. AM/AMBP-1 may be a novel treatment to attenuate the reperfusion injury after gut ischemia.

Allopurinol Prevents Erythrocyte Deformability Impairing but Not the Hematological Alterations After Limb Ischemia–Reperfusion in Rats

The measurement of red blood cell deformability provides a possible method for detecting the effect of ischemia–reperfusion on erythrocytes. In our study the effect of 1-h ischemia–reperfusion with or without allopurinol pretreatment on hematological parameters and red blood cell deformability was investigated in a follow-up experiment of 26 male CD outbred rats that were subjected to unilateral hind-limb ischemia by microvascular clips on femoral vessels for 1 h (IR, n = 6), some rats received allopurinol pretreatment under the same conditions (50 mg/kg, AP + IR, n = 8), others were subjected to sham operation (n = 6), and the rest of animals served as control (n = 6). Measurement of erythrocyte deformability using a bulk filtrometer with special setting of cell suspension hematocrit (1%), and determination of hematological parameters were performed daily for one week. In the IR group, relative cell transit time increased significantly on postoperative days 1 and 2, which was not observed in the other groups. Settings for the measurement of erythrocyte deformability by reducing the blood sample volume gave the possibility of monitoring the resulting changes in rats. Mean corpuscular volume and hemoglobin, platelet count, and platelet volume were higher in the IR and AP + IR groups than in the other groups. In summary, short-term ischemia and reperfusion induced lower red blood cell deformability in the early postoperative period, which could be prevented by allopurinol pretreatment.

Transient Cervical Nerve Root Compression in the Rat Induces Bilateral Forepaw Allodynia and Spinal Glial Activation: Mechanical Factors in Painful Neck Injuries

An in vivo rat model of transient cervical nerve root compression. To investigate the potential for cervical nerve root compression to produce behavioral hypersensitivity and examine its dependence on compression. Clinically, nerve root injury has been hypothesized as a potential source of neck pain, particularly because cervical nerve roots are at mechanical risk for injury during neck loading. Lumbar radiculopathy models of nerve root ligation show that mechanical allodynia and spinal glial changes depend on nerve root deformation magnitude. However, no investigation has been performed to examine cervical nerve root compression as a cause of pain. Two compressive forces (10 and 60 grams force [gf]) were transiently applied to the C7 nerve roots unilaterally using microvascular clips in separate groups (n = 12 each). Sham procedures were also performed in a separate group of rats (n = 12). Bilateral forepaw mechanical allodynia was monitored after surgery for 7 days. On day 7, spinal glial activation was assessed using immunohistochemistry to investigate its dependence on nerve root compressive force, in the context of behavioral hypersensitivity. Bilateral allodynia was observed following injury, which was significantly (P < 0.042) increased over sham and baseline responses. No difference in allodynia was found between the 10 and 60 gf injuries. Astrocytic and microglial activation were observed in the ipsilateral dorsal horn following compression, with only astrocytic activation paralleling allodynia patterns. Results imply a force threshold exists less than 10 gf for persistent pain symptoms following transient cervical nerve root compression. Findings also suggest that spinal glial activation may be related to behavioral sensitivity and may modulate cervical nerve root mediated pain.

Nitric oxide attenuates ischaemia-reperfusion (I/R) injury in the diabetic liver.

Liver ischaemia-reperfusion (I/R) occurs during resuscitation from haemorrhagic shock, hepatic transplantation and anatomic resection of the liver. This injury is associated with hepatocellular enzyme release and hepatocyte necrosis. The impact of chronic illnesses such as diabetes mellitus (DM) on hepatic I/R is unknown. This study determines the effect of DM on liver I/R using a murine model of type II DM in which the leptin receptor is defective. Preliminary studies suggest that animal models of DM have impaired endothelial nitric oxide (NO) release. Other studies suggest that NO attenuates hepatic I/R in phenotypically normal animals. We postulated that DM exacerbates hepatic I/R and that exogenous NO administration will attenuate hepatocellular injury. Non-diabetic and diabetic (db/db) mice were anaesthetized and underwent laparotomy with the placement of a microvascular clip on the hepatic artery and portal vein supplying the medial and left lateral lobes of the liver rendering about 70% of the liver ischaemic. Hepatic ischaemia was maintained for 45 min after which time the clip was removed and the liver segments reperfused. The abdomen was closed and the animals maintained for 5 h of reperfusion. Hepatic injury was then assessed by measuring serum alanine and aspartate transaminases (ALT, AST) spectrophotometrically. Sections of liver reperfused for 24 h were stained with haematoxylin and eosin and the percentage of hepatocyte necrosis evaluated using morphometric techniques. Other animals undergoing hepatic I/R received the NO donor (DETA 100 micro g/kg, i.v. 5 min prior to reperfusion). Time-matched, sham-operated animals served as controls. The data are expressed as mean +/- SEM and analysed by ANOVA. Serum AST and ALT levels were significantly higher in db/db animals vs. non-diabetics, even in the absence of hepatic I/R (P < 0.01). Serum AST and ALT levels in db/db mice undergoing hepatic I/R were nearly five times greater than that of non-diabetic animals (P < 0.01). Histologic examination of the livers of the diabetic animals undergoing I/R demonstrated significantly greater hepatocellular necrosis (zone III; 30-40%) when compared with non-diabetic animals sustaining the same injury (zone III; 3-10%). The NO donor DETA totally prevented the increase in serum ALT and AST release associated with I/R in both the diabetic and non-diabetic mice when compared with animals not receiving this agent (P < 0.01). This is the first study suggesting that DM exacerbates hepatic I/R and that NO donors will prevent this hepatocellular injury in the diabetic. Sixteen million Americans have DM. Understanding the effect of this chronic illness on the inflammatory response to injury is essential to improving clinical outcomes in these medically compromised patients.

Pharmacologic enhancement of rat skin flap survival with topical oleic acid.

This study was instituted to investigate in a rat model the effect of topical coadministration of the penetration enhancer oleic acid (10% by volume) and RIMSO-50 (medical grade dimethyl sulfoxide, 50% by volume) on rat skin flap survival. A rectangular abdominal skin flap (2.5 x 3 cm) was surgically elevated over the left abdomen in 40 nude rats. The vein of the flap's neurovascular pedicle was occluded by placement of a microvascular clip, and the flap was resutured with 4-0 Prolene to its adjacent skin. At the end of 8 hours, the distal edge of the flap was reincised to gain access to the clips and the clips were removed. After resuturing of the flap's distal edge to its adjacent skin, the 40 flaps were randomly divided into four groups. Group 1 (control) flaps were treated with 5 g of saline, group 2 (dimethyl sulfoxide) flaps were treated with 2.7 g of dimethyl sulfoxide (50% by volume), group 3 flaps (oleic acid) were topically treated with 0.45 g of oleic acid (10% by volume), and group 4 (dimethyl sulfoxide plus oleic acid) flaps were treated with a mixture of 0.45 g of oleic acid (10% by volume) and 2.7 g of dimethyl sulfoxide (50% by volume) diluted in saline. Each flap was topically treated with 5 ml of drug-soaked gauze for 1 hour immediately after clip removal to attenuate reperfusion injury. Thereafter, drug was applied topically once daily for 4 more days. Digital photographs of each flap were then taken on day 6 and the flaps were then harvested. The percentage of skin survival in each flap was determined by computerized morphometry and planimetry. The mean surviving area of group 3 (oleic acid-treated flaps) was 23.60 +/- 4.19 percent and was statistically higher than that in group 1 (control, saline-treated flaps) at 7.20 +/- 2.56 percent. The mean surviving area of group 2 (dimethyl sulfoxide-treated flaps) at 18.00 +/- 5.23 percent and group 4 (oleic acid- and dimethyl sulfoxide-treated flaps) at 9.90 +/- 3.44 percent did not achieve statistically higher mean surviving areas than controls. A topical solution of oleic acid (10% by volume) caused a statistically significant increase in the survival of rat abdominal skin flaps relative to controls. Dimethyl sulfoxide and the two experimental drugs together did not increase the percentage of flap survival when given as a single 5-ml dose released from a surgical sponge at reperfusion for 1 hour and then daily for a total of 5 days. The reasons for the lack of response are unknown but may have included the technical difficulty of delivering an adequate dose of dimethyl sulfoxide topically and immiscibility between dimethyl sulfoxide and oleic acid. Further studies may be warranted.

Changes in microcirculation after ischemic process in rat skeletal muscle.

Laser Doppler flowmetry (LDF) is a good method to investigate tissue microcirculation, but it has many standardization and measuring problems. To exclude these effects, we performed a test using LDF on rat skeletal muscle. In 12 CD outbred anesthetized rats, bilateral femoral vessels and the quadriceps femoris muscle were exposed. The left femoral artery and vein were clamped for 1 h by microvascular clips (ischemic side). On the right side, no other intervention was made (control side). An LDF probe was applied on the medial vastus muscle. Short-term occlusions (2-3 s) were performed before and after the 1-h clamping and on the control side while LDF curves were registered. The halftimes of ascending curves on the ischemic side were significantly elongated vs. the condition before clamping (P = 0.0007) or the control side (P = 0.0017). Microcirculatory changes caused by 1-h ischemia were shown by this simple, quick, and well-reproducible test on rat skeletal muscle.

Ischemic preconditioning reduces the severity of ischemia/reperfusion-induced pancreatitis

In various organs, including heart, kidneys, brain, liver and stomach, preconditioning by brief exposure to ischemia protects the organ against damage evoked by subsequent severe ischemia. This study has been undertaken to check whether two brief ischemic periods protect the pancreas against severe ischemia/reperfusion-induced pancreatitis and, if so, what is the role of sensory and vagal nerves in this phenomenon. In male Wistar rats, the ischemic preconditioning of the pancreas was performed by clamping of celiac artery (2×5 min with 5 min interval). Thirty minutes after preconditioning or sham operation, the ischemia/reperfusion-induced pancreatitis was evoked by clamping of inferior splenic artery for 30 min using microvascular clips, followed by 1 h reperfusion. Sensory nerves ablation was induced 10 days before final experiments by capsaicin. Truncal vagotomy was performed 1 week before the experiment. Exposure to regular 30-min pancreatic ischemia followed by 1 h reperfusion led to the development of acute hemorrhagic pancreatitis. Ischemic preconditioning, applied prior to induction of pancreatitis, caused the reduction in plasma lipase, plasma interleukin-1β and histological signs of pancreatic damage, as well as attenuated the reduction in pancreatic blood flow and DNA synthesis. Ablation of sensory nerves by capsaicin caused an aggravation of ischemia/reperfusion-induced pancreatic damage and attenuated a protective effect of ischemic preconditioning. Noxious effect of sensory nerves ablation on the pancreas was accompanied by the reduction in pancreatic blood flow and an increase in plasma interleukin-1β. Similar but less pronounced deleterious effect on the pancreas was observed after vagotomy. We conclude that: (1) pancreatic ischemic preconditioning reduces the severity of ischemia/reperfusion-induced pancreatitis; (2) this effect seems to be related, at least in part, to the improvement of pancreatic blood flow and the reduction in the release of proinflammatory interleukin-1β; (3) sensory and vagal nerves are involved in protective effect of ischemic preconditioning against pancreatic damage.

Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice

Problems with the intraluminal suture method for induction of focal cerebral ischemia in genetically altered mice include occurrence of subarachnoid hemorrhage (SAH) and variability of infarct volume. We hypothesized that use of 5-0 curved or 6-0 straight suture for carotid cannulation might decrease SAH and that the application of a microvascular clip to the common carotid artery (CCA) might decrease variability of infarct volume. The purpose of this study is to evaluate and explain the results of these technical modifications. Strain related differences in vascular anatomy were evaluated. Male C57BL/6 mice were divided into two groups for permanent and temporary middle cerebral artery occlusion (MCAO). Results of 5-0 curved suture and 6-0 straight suture insertion with and without CCA clip application were examined. Cerebral perfusion was monitored by laser-Doppler flowmetry and infarct volume was measured. After permanent MCAO, larger and more consistent infarct volumes resulted using CCA clip application with a 6-0 but not with a 5-0 suture. After temporary MCAO, the SAH rate was 12.5% with a 5-0 curved suture and 11.1% with a 6-0 straight suture. A 40% rate was observed in a pilot study with 5-0 straight suture. Infarct volume after temporary MCAO with a CCA clip was significantly larger and variability of infarct volume was smaller than without the CCA clip using 5-0 curved and 6-0 straight suture. In summary, SAH is less frequent using a 5-0 curved or 6-0 straight suture. Infarct volume is enlarged by application of a CCA clip (249).

Effects of recombinant tissue plasminogen activator after intraluminal thread occlusion in mice: role of hemodynamic alterations.

It has been suggested that recombinant tissue plasminogen activator (rtPA) may cause an aggravation of injury after transient focal ischemia via excitotoxic side effects. Such rtPA toxicity would be of major clinical significance since rtPA is increasingly used in stroke treatment. This study was conducted to evaluate the effects of dose, application time, and hemodynamic changes after intravenous rtPA treatment in focal ischemia. Mice were subjected to a 90-minute episode of middle cerebral artery thread occlusion, and rtPA effects were assessed by laser-Doppler flowmetry, [(14)C]iodoantipyrine autoradiography, and triphenyltetrazolium chloride staining. We provide evidence that rtPA provokes complex hemodynamic alterations in the ischemic brain tissue, which include an initial hyperperfusion and a more delayed hypoperfusion response. Changes are most pronounced in the periphery of the ischemic infarct, where regional blood flow drops below critical thresholds of tissue viability. Our observations suggest that changes of perfusion may at least partly explain the rtPA-induced increase of infarct size, which has previously been reported and which we also confirmed in the present experiments. Notably, both the secondary hypoperfusion and increase of infarct volume were abolished when rtPA-treated animals received additional heparin infusions. This finding suggests that a secondary hypercoagulability may compromise brain perfusion after rtPA delivery. Accordingly, early treatment with heparin might help to prevent the rtPA-induced changes.

Postischemic Morphological Changes of the Dentate Gyrus in the Gerbil During Reperfusion

Postischemic lesion following reperfusion have been investigated in the gerbil by temporary occlusion of a common carotid artery. Two phenomena have been reported in injured ischemic cells: the inability to restore mitochondrial function and evidence of plasma membrane damage. For Further understanding the pathophysiological mechanism of ischemic lesion, we have evaluated the various ischemic period and early postischemic reperfusion. in addition to the morphological changes of the dentate gyrus by light microscopy, we employed the immunocytochemical staining for iNOS and eNOS.The mongolian gerbil, Meriones unguiculatus, were anesthetized with α-chloralose (350mg/kg), bilateral common carotid artery occlusion was induced for 90 minutes. The right common carotid artery was ligated with 5-0 silk suture, and the left was clamped by a micro vascular/miniature aneurysmal clip. After an ischemic period of 90 minutes, restoration of the blood flow of the left common carotid artery was accomplished by withdrawing the microvascular clip.

The role of melatonin in prevention of intestinal ischemia-reperfusion injury in rats

Background/Purpose: The aim of this study was to determine the effect of melatonin, a hormone that is known as an antioxidant, on the prevention of tissue damage during mesenteric ischemia/reperfusion (I/R). Methods: A total of 40 young Wistar-albino rats were divided equally into 4 groups with varied treatment. Group 1 was control (sham), group 2 was I/R, group 3 was I/R plus melatonin (10 mg/kg) and group 4 was I/R plus melatonin (20 mg/kg). I/R was realized as follows: after laparatomy, a microvascular atraumatic clip was placed across the superior mesenteric artery (SMA) under general anaesthesia, and it was removed after ischemia for 30 minutes. The first dose of melatonin was applied intraperitoneally at the start of reperfusion. The second and third doses were applied intramuscularly on the first and second day. Only SMA dissection under general anaesthesia was carried out in the control group rats. On the third day of the study all the rats were killed, and their bowels were removed. Malondialdehyde (MDA) levels were assayed as an index of lipid peroxidation reflecting free radical reaction in the intestine. Histopathologic analysis was made using light microscopy in a blind fashion. Results: The levels of tissue MDA were found to be significantly lower in groups 3 and 4 compared with group 2 (P <.05). The MDA levels of group 4 did not differ significantly from that of the control group (P >.05). The histopathologic results were consistent with the MDA levels. Conclusion: These results suggest that melatonin has a strong antioxidant effect in preventing intestinal I/R damage, and that this effect is exerted in a dose-dependent manner. J Pediatr Surg 35:1444-1448. Copyright © 2000 by W.B. Saunders Company.

Recombinant human hepatocyte growth factor protects the liver against hepatic ischemia and reperfusion injury in rats.

Recent evidence indicates that hepatocyte growth factor (HGF) has a cytoprotective effect against hepatic injury caused by hepatotoxins and inflammatory cytokines. Studies were performed to determine whether HGF influences the survival rate of rats subjected to hepatic warm ischemia/reperfusion (WI/Rp) injury. Male Sprague-Dawley rats were subjected to total or segmental hepatic ischemia by occluding the hepatic artery, portal vein, and bile duct with a microvascular clip. Rats were treated with four intravenous injections of recombinant human HGF (rhHGF 1 mg/kg) or the vehicle 72, 48, 24, and 12 h before surgery. None of the eight animals in the control group were alive 12 h after total hepatic WI/Rp. Seven of eight animals in the rhHGF-treated group were alive more than 2 days after the reperfusion. In the model of segmental hepatic ischemia, rhHGF inhibited the increase in cytokine-induced neutrophil chemoattractant in serum. The number of neutrophils infiltrating the liver was significantly lower in the rhHGF-treated group than in the control group. rhHGF prevented increases in the activity of serum alanine transaminase and in hepatic necrosis. Experiments with proliferating cell nuclear antigen staining demonstrated that hepatocyte proliferation markedly increased in rhHGF-treated rats as compared with controls. These results indicate that HGF facilitates recovery of the liver from hepatic WI/Rp injury, at least in part through the prevention of neutrophil infiltration and the activation of hepatocyte proliferation.

Spinal Cord Stimulation Improves Survival in Ischemic Skin Flaps: An Experimental Study of the Possible Mediation by Calcitonin Gene-Related Peptide

Currently, spinal cord stimulation is used to treat ischemia and ischemic pain, with the best results observed in vasospastic cases. It was earlier demonstrated that spinal cord stimulation may attenuate experimentally induced vasospasm in an island flap in the rat. The present study was designed to investigate whether preemptive spinal cord stimulation could increase long-term flap survival and to explore the neurohumoral mediation of the effect. A total of 56 rats were implanted with chronic spinal cord stimulation systems. Three days later, a groin flap based on the superficial epigastric vessels was harvested, and the single feeding artery was occluded by a detachable microvascular clip. After 12 hours, the clip was removed. Flap survival was evaluated after 7 days. Immediately before flap surgery, two groups of animals received 30 minutes of stimulation using current clinical parameters and with stimulation amplitudes of 70 (n = 10) or 90 percent (n = 8) of that evoking muscular contractions. The outcomes in these groups were compared with those in two control groups (n = 20; n = 10). In one group, an additional calcitonin gene-receptor peptide (CGRP) antagonist was intravenously injected before stimulation (n = 8). In the control groups without stimulation, virtually all flaps necrotized. In treated groups, flap survival was 60 percent at the lower intensity and almost 90 percent at the higher one. The administration of a CGRP antagonist before treatment reduced its efficacy to below 40 percent survival. The differences between the untreated and treated groups were significant. The decrease in survival after CGRP-receptor block was significant in one of two tests. Preemptive spinal cord stimulation increases survival of skin flaps with critical ischemia. The effects are dependent on the stimulation intensity and are possibly mediated by the release of CGRP in the periphery.

Spinal Cord Stimulation Improves Survival in Ischemic Skin Flaps: An Experimental Study of the Possible Mediation by Calcitonin Gene-Related Peptide

Currently, spinal cord stimulation is used to treat ischemia and ischemic pain, with the best results observed in vasospastic cases. It was earlier demonstrated that spinal cord stimulation may attenuate experimentally induced vasospasm in an island flap in the rat. The present study was designed to investigate whether preemptive spinal cord stimulation could increase long-term flap survival and to explore the neurohumoral mediation of the effect. A total of 56 rats were implanted with chronic spinal cord stimulation systems. Three days later, a groin flap based on the superficial epigastric vessels was harvested, and the single feeding artery was occluded by a detachable microvascular clip. After 12 hours, the clip was removed. Flap survival was evaluated after 7 days. Immediately before flap surgery, two groups of animals received 30 minutes of stimulation using current clinical parameters and with stimulation amplitudes of 70 (n = 10) or 90 percent (n = 8) of that evoking muscular contractions. The outcomes in these groups were compared with those in two control groups (n = 20; n = 10). In one group, an additional calcitonin gene-receptor peptide (CGRP) antagonist was intravenously injected before stimulation (n = 8). In the control groups without stimulation, virtually all flaps necrotized. In treated groups, flap survival was 60 percent at the lower intensity and almost 90 percent at the higher one. The administration of a CGRP antagonist before treatment reduced its efficacy to below 40 percent survival. The differences between the untreated and treated groups were significant. The decrease in survival after CGRP-receptor block was significant in one of two tests. Preemptive spinal cord stimulation increases survival of skin flaps with critical ischemia. The effects are dependent on the stimulation intensity and are possibly mediated by the release of CGRP in the periphery. (Plast. Reconstr. Surg. 103: 1221, 1999.)

Carbonyl histochemistry in rat reperfusion nerve injury

Free radical mediated, site-specific lipid and protein oxidation has been implicated in the pathophysiology of an ischaemic/reperfusion injury. The aim of the present study was to determine whether carbonyl formation could be detected histochemically in reperfused rat sciatic nerves. We also examined the effects of preischaemic α-tocopherol supplementation on carbonyl formation in reperfused nerves. Seven hours of near-complete ischaemia was induced in rat right hindlimb by occlusion of major arteries using microvascular clips. Histochemical detection of carbonyl compounds, applying naphthoic acid hydrazide (NAH) and Fast Blue B (FBB), was undertaken at thigh, knee and calf levels of sciatic, tibial and peroneal nerves. NAH–FBB reactivity was confined to vessels in reperfused nerves. Positively stained epi-, peri- and endoneurial vessels were invariably observed after 2 h of reperfusion at all levels examined. After 24 and 48 h and 7 days of reperfusion, NAH–FBB-positive vessels were more frequently found at knee and calf levels than at the thigh level. Following preischaemic α-tocopherol supplementation, no vessels were stained positively with NAH–FBB, except for some epineurial vessels at knee and calf levels after 2 h of reperfusion. Morphometry in endoneurial vessels at the knee level revealed that endothelial cell area in α-tocopherol-treated reperfused nerves was significantly less when compared with those in reperfused nerves without α-tocopherol. In conclusion, we have demonstrated histochemical evidence of carbonyl formation in vessels, but not with nerve fibres, in ischaemic/reperfused rat sciatic nerves. These abnormalities were prevented with preischaemic supplementation of α-tocopherol.

Pretreatment with activated protein C or active human urinary thrombomodulin attenuates the production of cytokine-induced neutrophil chemoattractant following ischemia/reperfusion in rat liver

We investigated the role of anticoagulant in the ischemia/reperfusion injury of the liver, using activated protein C (APC), active human urinary thrombomodulin (UTM), and factor Xa blocked at the active site (DEGR-Xa). Liver ischemia was induced in male Wistar rats by occlusion of the portal vein with a microvascular clip for 30 minutes. Each anticoagulant was injected intravenously 10 minutes before clamping the portal vein. Serum concentrations of cytokine-induced neutrophil chemoattractant (CINC) were determined by enzyme-linked immunosorbent assay. The serum levels of CINC increased significantly following reperfusion, reaching a peak in 6 hours, and then decreasing gradually to control levels by 24 hours. CINC levels in rats pretreated with APC (500 U/kg), UTM (3,000 TMU/kg), or DEGR-Xa (10 mg/kg) peaked 3 hours following reperfusion and decreased rapidly to baseline level within 6 and 12 hours, respectively. These peak values were significantly lower than those observed in untreated rats (P < .01). Expression of CINC transcripts in liver tissue of untreated rats was evaluated by Northern blot analysis and peaked 3 hours following reperfusion. Pretreatment with these anticoagulants significantly decreased the expression of CINC messenger RNA transcripts as compared with untreated animals. Myeloperoxidase activity and the number of neutrophils accumulated into the liver 24 hours following ischemia/reperfusion were also significantly decreased in animals pretreated with these anticoagulants. In addition, correlations between the peak values of liver enzymes and serum CINC levels were found to be significant (P < .001). The inactive derivative of factor Xa, a selective inhibitor of thrombin generation, inhibited ischemia/reperfusion-induced increases in the serum concentration and messenger RNA transcript quantities of CINC. The inactive factor Xa also reduced hepatic accumulation of neutrophils after ischemia/reperfusion. These results indicate that the release of CINC is likely related to the hepatic microcirculation disturbance induced by microthrombotic occlusion following ischemia/reperfusion.

Early phase alterations in endothelium dependent vasorelaxation responses due to aneurysm clip application and related manipulations.

Mechanically induced vasoconstriction observed throughout surgery and in the immediate postoperative period was investigated to assess the effects of various microsurgical manipulations. Factors such as the type of aneurysm clip, duration of temporary clipping and peri-adventitial tissue stripping were the variables in this study. Microsurgical clips were applied on guinea pig "cervical carotid arteries" in which peri-adventitia had been removed microsurgically. Arterial rings were removed immediately after surgery. Endothelium dependent relaxations were measured and morphological investigations were performed using light microscopy. It was observed that as the clip application period increased, relaxation responses decreased. Peri-adventitial tissue stripping caused a marked decrease in the relaxation responses in all types of the clips. Microvascular clips, in spite of their lower closing forces, had the greatest deleterious effect on relaxation responses of the vessel, in both normal and peri-adventitial tissue stripped. When the peri-adventitial tissue of the vessel had been stripped, convolutions of the lamina elastica interna were found to be lost in parallel with the decreased tonus of the artery. In the vessels subjected to clipping endothelial denudation and cracking took place. As a conclusion it can be stated that both peri-adventitial tissue stripping and microvascular clip application have deleterious effects in the early postoperative period. While choosing clips from minimal occlusion force tables, care must be taken to choose clips with less width; and while performing microvascular anastomosis, temporary clips with a lesser width must be used in place of microvascular clips. Adventitial stripping must not be unnecessarily generous during microvascular anastomosis.

Neuroprotective efficacy of lifarizine (RS-87476) in a simplified rat survival model of 2 vessel occlusion.

1. A new, modified rat two vessel occlusion model (with hypotension) was established and the neuroprotective efficacy of the novel agent lifarizine (RS-87476) was examined. 2. The two vessel occlusion model used in the study was a modification of the model described in the literature, whereby we have obviated the need to use a muscle relaxant and intubate the trachea to provide ventilatory support by providing a tight fitting face mask attached to the ventilator. Furthermore, the need to combine exsanguination and additional pharmacological means of inducing the mandatory hypotension (50 mmHg), required to decrease brain blood perfusion pressure, has been removed by simply manipulating the concentration of the already present halothane anaesthetic. 3. The appropriate level of hypotension having been reached, microvascular clips were applied to bilaterally occlude the common carotid arteries for 12 min. This resulted in a loss of the cortical EEG activity. Local cerebral blood flow was measured 6 min into the occlusion period, using the fully quantitative [14C]-iodoantipyrine autoradiographic technique, in a separate group of rats (n = 5). This illustrated the lack of any blood flow, in the areas under study, during the period when there was an isoelectric cortical EEG pattern. 4. The high grade global ischaemic lesion which occurred gave quantifiable neuronal damage in several vulnerable regions of the brain, namely, the hippocampal CA1 sub-field, cortex, thalamus, striatum, and cerebellar brain stem (Purkinje cells). 5. Following the global ischaemic insult the rats were allowed to recover for 72 h before assessment of the damage, during which time one group of rats (n = 11) received 100 micrograms kg-1 lifarizine i.a. 5 min post-occlusion, 500 micrograms kg-1 lifarizine i.p. 15 min post-occlusion, and 500 micrograms kg-1 lifarizine i.p. twice daily for 72 h. A second group of rats (n = 12) was treated with appropriate volumes of vehicle (0.4 ml kg-1 i.a. and 2 ml kg-1 i.p.) at identical time points. 6. Histopathological damage was assessed, from cresyl violet and haematoxyline/eosin stained sections, using a scoring system of 0-6 (no damage-complete neuronal death). The dosing regimen of lifarizine gave reduced damage in the hippocampal CA1 sub-field (4.1 +/- 0.3 to 2.8 +/- 0.6) and striatum (1.7 +/- 0.3 to 1.2 +/- 0.3) and significant neuroprotection in the anterior cortex (2.0 +/- 0.2 to 1.2 +/- 0.2; p < 0.05), thalamus (1.5 +/- 0.2 to 0.8 +/- 0.2; p < 0.01), posterior cortex (1.5 +/- 0.2 to 1.0 +/- 0.2; p < 0.05) and cerebellar brain stem (0.9 +/- 0.2 to 0.4 +/- 0.1; p < 0.01). The overall mean brain score was significantly reduced (from 1.5 +/- 0.1 to 0.9 +/- 0.2). 7. These data show that the newly modified 2 vessel occlusion model produced a quantifiable level of ischaemic damage and that the novel agent lifarizine is neuroprotective in the model.