Long-En Chen

Assessment of ischemia and reperfusion injury

Direct videomicroscopy of the rat cremaster muscle microcirculation supplemented by animal models of replantation, vascular crushing, and muscle function after injury and recovery were used to investigate the occurrence of reperfusion failure. It is evident that failure of blood reflow may be induced by multiple factors that can be grouped into categories of ischemia, intimal damage, and systemic or local responses, which are referred to as the no reflow triad. The components comprising the 3 sides of the no reflow triad can interact with one another in an intricate manner, and any single factor or combination of factors is capable of triggering the events leading to reperfusion failure. The pronounced regional nature of reperfusion injury and the direct relationship between the severity of the observed vascular alterations and increasing duration of ischemia have been documented. The dynamic changes and histopathology of the microcirculation included constriction of the arteries, swelling of endothelial and leukocytes, and erythrocyte rouleaux formation during ischemia. As ischemia duration was lengthened, the degree of these changes increased correspondingly. The changes on reperfusion were disruption of blood flow patterns, vortex formation, regional stasis, adhesion and migration of leukocytes, focal hemorrhage, edema, vasospasm, and platelet aggregation. The deleterious effects of systemic acidosis, interstitial hemorrhage, denervation, and prolonged venous occlusion were subsequently documented. The application of information gained from this series of laboratory experiments has resulted in continued improvement in the success rate in clinical microvascular surgery.

Recombinant human glial growth factor 2 (rhGGF 2) improves functional recovery of crushed peripheral nerve (a double-blind study)

This in vivo double-blind study evaluated the effect of recombinant human glial growth factor 2 (rhGGF2), a Schwann cell mitogen, on the recovery of motor function of rat sciatic nerve following crush injury. Seventy three rats were divided into three groups. Group I (n=5), sham operated; Groups II (n=34) and III (n=34) received a 100 g crush load for 2 h over a 5 mm segment of the sciatic nerve. Group III was treated with 1 mg/kg rhGGF2, via subcutaneous injection one day before nerve crush and daily for the following four days. Group II received an equivalent volume of saline as a control. Motor functional recovery was assessed by calculating the sciatic functional index (SFI) and the recovery rate of tetanic contractile force of the extensor digitorum longus (EDL) muscle. Recovery of nerve function was evident at day 11 after crush in the rhGGF2-treated animals, whereas the nerves in controls were still paralyzed. The rhGGF2-treated animals showed a significant improvement of the SFI between days 11-21 postoperatively when compared to controls. The isometric tetanic contractile force was stronger in the rhGGF2-treated group than in controls, with a significant difference at 40 to 70 Hz stimulus frequencies on day 4. Correlation analysis showed that tetanic contractile force had a linear correlation with the SFI. Histologic assessment indicated that the rhGGF2-treated animals showed less severe degeneration and earlier robust remyelination of axons than controls. The results suggest that treatment with rhGGF2 is effective in promoting nerve regeneration as seen in measurements of functional recovery and qualitative assessment of nerve morphology. The mechanism of GGFs protective effect may be related to its direct action on Schwann cells, stimulating their mitosis as well as inducing neurotrophic factors essential to neuronal maintenance and repair.

Pathophysiology and related studies of the no reflow phenomenon in skeletal muscle.

Although the success rate of microvascular replantation and revascularization procedures has increased steadily since the 1960s, some replanted tissues do not reperfuse despite technically adequate arterial anastomoses. This failure of microvascular perfusion is termed no reflow. Much research has been directed toward discovering the etiology of no reflow since it was first described 25 years ago. Three pathophysiologic processes have been identified as playing a central role in the development of no reflow: intracellular calcium overload, oxygen-free radical medicated damage, and altered arachidonic acid metabolism. The first tissue believed to be injured irreversibly by these processes is the endothelium, which leads to dysfunction of the parenchymal cells. All 3 pathways are interrelated extensively, which allows for pharmacologic intervention at many different steps. Agents that have been shown to be beneficial in preventing no reflow include calcium channel blockers, prostaglandin analogs, thromboxane synthesis inhibitors, vasodilators, thrombolytics, and many antioxidants. Although they have been shown to be effective in various laboratory models, additional investigation is necessary before these treatments can be established in clinical use.

S-nitroso-N-acetylcysteine protects skeletal muscle against reperfusion injury

The effects of a nitric oxide (NO) donor on microcirculation and contractile function of reperfused skeletal muscle were studied. Rat cremaster muscles underwent 5 hours of ischemia and 90 minutes of reperfusion and were divided into two groups systemically infused with S‐nitroso‐N‐acetylcysteine (SNAC, 100 nmol/ min) and phosphate‐buffered saline (PBS), respectively. The results showed that the vessels in the SNAC group had more rapid and complete recovery than that in controls. A significant difference was found from 10 to 40 minutes and at 90 minutes in 10–20‐μm arterioles, from 10 to 90 minutes in 20–40‐μm arterioles, and at 10 and 90 minutes in 40–70‐μm arteries. When compared to controls, SNAC‐treated muscles showed larger fluorescein filling areas at 15, 30, 60, and 90 minutes and greater isometric tetanic contractile forces in response to stimulation frequencies of 40, 70, 100, and 120 Hz. The data indicate that supplementation of exogenous NO could effectively improve microcirculation and contractile function of skeletal muscle during early reperfusion.

Antithrombotic potencies of enoxaparin in microvascular surgery: Influence of dose and administration methods on patency rate of crushed arterial anastomoses

This study evaluated the influence of the dose and administration methods of enoxaparin, a low-molecular-weight heparin, on the patency rate of crushed rat femoral arteries following anastomosis. An impact crush with a 25-kg magnitude was applied to a 2-mm segment of 100 rat femoral arteries, followed by anastomosis. The arteries were divided into five groups: group 1 received systemic enoxaparin alone with a relatively high dose (45 IU) twice a day for 3 days; groups 2 and 3 received topical irrigation with a lower (15 IU/mL) concentration and a higher (45 IU/mL) concentration, respectively; group 4 received systemic and topical application at a lower (15 IU) dose and concentration (15 IU/mL); and group 5 received systemic and topical application at a higher (45 IU) dose and concentration (45 IU/mL). The results of this study demonstrate the following: (1) topical irrigation with enoxaparin at a concentration of 45 IU/mL-three times higher than that recommended for clinical use adjusted by body weight (15 IU/mL)-is effective for antithrombotic action; (2) a combination of systemic and local application does not offer additional benefit in the patency rate when compared to local irrigation alone; (3) systemic administration alone does not prevent thrombus formation; and (4) enoxaparin is potentially useful to enhance the patency rate in compromised microvessels.

Functional effects of lymphotoxin on crushed peripheral nerve

The effect of lymphotoxin (LT) on the functional recovery of crushed peripheral nerves was studied. Using a specially designed compression device, a 5 mm segment of the right sciatic nerve of rats was subjected to a 100 g crush load with a 2 hr duration. The rats in the experimental and control groups received two doses of LT (20 μg/kg each) or the same volume of saline, respectively, administered intraperitoneously 24 hr and 1 hr before the procedure. Walking track tests and histologic examinations were performed at intervals up to 56 days after the crush. Motor functional recovery in the LT pretreated group started at day 7 while the crushed limb in the control group remained totally dysfunctional. The sciatic functional index improved faster in the LT group than in the control group during the second week after the crush and reached a significant difference (P < 0.05) at day 18. Subsequently, both groups had a similar evolution. Histologic results paralleled the functional findings. In conclusion, LT can promote motor functional recovery of crushed rat peripheral nerve in the early stage of regeneration.

Vasodilator Action of Pentoxifylline on Microcirculation of Rat Cremaster Muscle

Pentoxifylline improves microvascular blood flow in conditions of vascular insufficiency. The clinical benefits of pentoxifylline have been attributed to its effects on the cellular elements of whole blood, although a few studies suggest it may also be a vasodilator. The purpose of this study was to determine whether pentoxifylline has a vasodilator effect on the luminal diameter of small arteries preconstricted with norepinephrine and on resting small arteries in the rat cre master muscle. Intravital videomicroscopy was used in order to observe directly the vasodi lator capacity of topically applied pentoxifylline. The results reveal that pentox ifylline (100 μM and more) can significantly dilate small arteries preconstricted with norepinephrine. Pentoxifylline had no effect on the diameter of resting small arteries. These results suggest that vasodilation may play a role in the ability of pen toxifylline to improve arterial blood flow.

Effects of dexamethasone on the contractile function of reperfused skeletal muscle

This study evaluated the effects of dexamethasone (DXM) on contractile function of reperfused extensor digitalis longus (EDL) muscles following 3‐hour ischemia and 24‐hour reperfusion. The rats were divided into four groups: normal muscle, ischemia with saline treatment, ischemia/reperfusion with saline treatment, and ischemia/reperfusion with DXM treatment groups. DXM (0.6 mg kg−1) or saline (3.0 ml kg−1) was administered at 3 hours prior to ischemia. Results showed that although contractile force in all three treated groups was significantly lower than that of normal EDL, the average isometric tetanic contractile force in the DXM‐treated group was significantly greater than that in the saline‐treated ischemia and ischemia/reperfusion groups. A significant difference was also seen at the peak force and at 5 seconds of the fatigue trains, and with a longer fatigue half‐time (FT1/2) in the DXM‐treated group than in the other two groups. Histologically, edema, inflammation and necrosis of muscle fiber were less severe in the DXM‐treated group than in the saline‐treated group. The results indicate that pretreatment with DXM appears to attenuate, but does not completely reverse, the contractile function deficit of ischemic skeletal muscle during the first 24 hours of reperfusion. MICROSURGERY 17:313–320 1996

Actions of glucocorticosteroids on ischemic-reperfused muscle and cutaneous tissue

Ischemia‐reperfusion injury represents a complex series of vascular and cellular events that resembles an acute inflammatory reaction within the reperfused tissue. This article provides an overview of glucocorticosteroid effects on cells and tissues involved in inflammatory reaction following ischemia‐reperfusion of muscle and cutaneous tissue. Glucocorticosteroids exert a variety of effects that influence the microcirculation. These effects include leukocyte recruitment, reduction of vascular permeability, inhibition of formation of cytokines or other mediators, and modulation of enzyme systems involved in inflammation. The current view is that glucocorticosteroids act through cytoplasmic receptors by controlling the transcription of certain genes encoding regulatory proteins, but the exact mechanisms of glucocorticoid action on ischemia‐reperfusion are not completely understood. Potential mechanisms may involve modulation of neutrophil and endothelial function, inhibition of formation of arachidonic acid products, and attenuation of lipid peroxidation of biological membranes through membrane stabilization and scavenging of toxic free radicals

iNOS inhibitor 1400W improves microcirculation of denervated cremaster muscle following ischemia/ reperfusion

Introduction: Nitric oxide (NO) may play a predominant role in ischemia/reperfusion (I/R) injury, both in microcirculation and function. This study was designed to observe the effects of the selective inducible NO synthase (iNOS) inhibitor, 1400W, on the microcirculation of denervated I/R skeletal muscles. Methods: Under anesthesia, the left cremaster muscles of 96 rats weighing 90 ‐110 g were isolated, opened, denervated, and spread onto a transparent acrylic microscope stage. 1400W was administered subcutaneously 10 min prior to reperfusion. Results: Following 3 hrs of ischemia, the mean blood flow of the muscle measured by using laser Doppler flowmetry was 30.2 6 16% of baseline in controls at 10 min and gradually increased to 67.4 6 32% by the end of 90 min reperfusion. In the 1400W group, mean blood flow reached 76.3 6 14% at 10 min and a maximum of 106.3% 6 18% at 50 min of reperfusion, with a significantly (p , 0.01) greater flow than those in controls at all time points with the exclusion of that at 60 and 90 min. Intravital microscopy examination showed the average diameters of 10 ‐70 mm arteries in controls were between 54% and 62% of baseline at 10 min and gradually increased to a maximum of 79 6 11% in 10 ‐20 mm, 73 6 14% in 21‐ 40 mm, and 85 6 3% in 41‐70 mm arteries at 90 min of reperfusion. In contrast, the diameter of 3 vessel categories in the 1400W group sharply increased to over baseline at 10 min and remained at this level throughout 90 min of observation. There was a significant (p , 0.001) difference in mean vessel diameter between the two groups at every time point. Data analyzed using ANOVA. Conclusions: 1) I/R results in an increase of iNOS activity and/or production in the reperfused tissues. 2) Selective inhibition of iNOS improves microcirculation in the reperfused tissue, thereby reducing I/R injury and improving the ‘no-reflow‘ phenomenon. Our data support an iNOS-mediated mechanism in I/R injury and indicate that the inhibition of iNOS in I/R injured muscle could have potential clinical benefit.