Valeri S. Chekanov

Transplantation of autologous endothelial cells induces angiogenesis.

CHEKANOV, V., et al.: Transplantation of Autologous Endothelial Cells Induces Angiogenesis. This study examined the feasibility and efficacy of autologous endothelial cell (EC) transplantation using a fibrin matrix in the ischemic myocardium of sheep. Four weeks after placing an ameroid constrictor in the circumflex artery of 12 adult sheep, four animals (EC group) were subjected to EC transplantation. In four others (saline [SAL] group) saline with added inactivated cells was injected and four animals served as controls. Eight weeks after treatment the animals were sacrificed to assess histology and ultrastructure. Eight weeks after injection, ventricular function was markedly improved in the EC transplant group, but had deteriorated in the SAL and control groups. Myocardial blood flow was also increased in the EC group. Histology and electron microscopy revealed extensive neovascularization after EC transplantation and improved myocardial appearance. Heterotopic transplantation of EC within a fibrin matrix enhances neovascularization, increases myocardial blood flow, and improves left ventricular function. (PACE 2003; 26[Pt. II]:496–499)

Validation of a left atrial lesion pattern for intraoperative ablation of atrial fibrillation

BACKGROUND Evidence that atrial fibrillation may begin in early stages from triggers or reentry circuits primarily in the left atrium suggests that the entire Maze 3 lesion pattern may be unnecessary. In the present study we describe a new left atrial lesion pattern for intraoperative linear ablation of chronic atrial fibrillation. METHODS Endocardial radiofrequency ablation was performed on 12 dogs with chronic atrial fibrillation. Lesions to isolate pulmonary veins in pairs, the left atrial appendage, and connecting lesions between these structures were administered in a randomized approach. RESULTS Twelve dogs were in chronic atrial fibrillation for 31 +/- 21 days before ablation. Atrial fibrillation was successfully ablated and rendered noninducible in all 12 dogs. All treatment failures observed with less than the full lesion pattern became a success when the remaining lesions were given. CONCLUSIONS Atrial fibrillation ablation using this left atrial lesion pattern is highly successful in this model. This approach may have significant utility as a concomitant procedure for patients with atrial fibrillation undergoing mitral valve procedures.

Deferoxamine enhances neovascularization and recovery of ischemic skeletal muscle in an experimental sheep model.

BACKGROUND Iron chelators have been reported to interfere with inflammatory cells and possibly enhance vascular growth factor expression. The objective of this study was to investigate the efficacy of the iron chelator deferoxamine mesylate in preventing skeletal muscle ischemia. METHODS The latissimus dorsi muscle (LDM) was mobilized in 20 adult sheep. Two separate pockets were created in each sheep. Autologous fibrin sealant with or without 100 mg/mL of deferoxamine mesylate (10 pockets) was added to the pockets. Deferoxamine mesylate alone was also applied to another 10 pockets, whereas the 10 other pockets served as controls. RESULTS Conventional, indirect immunofluorescent enface staining showed that in nonmobilized, nonischemic LDM the capillary density was 196 +/- 14 capillaries/mm2 in the distal and 207 +/- 19 capillaries/mm2 in the middle part. After severe ischemic shock (subtotal mobilization), the muscle did not recover completely even after 2 months (149 +/- 15 capillaries/mm2 in the distal part and 177 +/- 16 capillaries/mm2 in the middle part of the LDM). Fibrin application only increased muscle neovascularization. The number of capillaries per mm2 of muscle increased to 250 +/- 25 in the distal part and to 271 +/- 24 in the middle part of the LDM. However, when fibrin was applied with added deferoxamine mesylate, the capillary density increased to 361 +/- 25 capillaries/mm2 in the distal part (p < 0.05 vs fibrin only; controls) and to 401 +/- 20 capillaries/mm2 in the middle part of the LDM (p < 0.05 vs fibrin only and p < 0.001 vs controls). The data are concordant with the blood flow estimation before and after mobilization (severe ischemic shock) in the different parts of the LDM. CONCLUSIONS Local application of deferoxamine mesylate enhances neovascularization and recovery of surgically induced skeletal muscle ischemia in a sheep model.

Direct fibrin injection to promote new collateral growth in hind limb ischemia in a rabbit model.

Abstract Local stimulation of angiogenesis is a new approach for the treatment of critical limb ischemia. Our investigation tested intramuscular (IM) injection of a modified fibrin meshwork in a rabbit model. Methods: The left external iliac and femoral arteries were excised in 24 rabbits that were divided into four groups: control; IM saline injection; fibrin meshwork plus low dose (2.5 mg) fibrinogen IM; fibrin meshwork plus high‐dose (5.0 mg) fibrinogen IM. Angiography was performed before surgery, immediately after surgery, and one month postoperatively. Lower limb‐calf blood pressure was measured immediately after surgery and at postoperative days 10, 20, and 30. On day 30, conventional indirect immunostaining was performed to determine the percentage of the area occupied by capillaries. Results: Immediately after surgery, in all four groups, the number of contract‐opacified arteries (COA) crossing a specific segment of a grid decreased from 5.3 ± 1.3 to 3.2 ± 1.0 (p < 0.05) ; the number of grid intersections decreased from 30.2 ± 6.5 to 19.3 ± 4.8 (p < 0.05) ; and the total number of grids with COA decreased from 18.3 ± 3.8 to 12.2 ± 2.5 (p < 0.05) . One month after surgery, in the control group, these parameters were 6.2 ± 1.1, 33.2 ± 5.7 and 20.3 ± 1.5 , respectively; in the saline‐treated group, these parameters were 6.1 ± 0.8, 28.3 ± 6.9 and 19.8 ± 1.1 , respectively ( p > 0.05 versus control and versus baseline data). When fibrin containing 5.0 mg fibrinogen was used, these parameters increased to 8.5 ± 0.9, 48.3 ± 5.1 , and 27.1 ± 0.9 , respectively ( p < 0.001 versus immediately after surgery and p < 0.05 versus control). In all four series, no Doppler flow signal was detected from the posterior tibial artery by day 10. By day 30, the lower limb‐calf blood pressure ratio had improved in all four series, but was significantly improved in only the two groups treated with fibrin sealant ( 0.3 ± 0.05 control; 0.3 ± 0.08 saline; 0.6 ± 0.06 fibrinogen 2.5; 0.7 ± 0.05 fibrinogen 5.0). Conclusion: Intramuscular injection of a fibrin meshwork considerably increased angiogenesis in the severely ischemic hind limb and may be strongly recommended for clinical use in patients with limb‐threatening ischemia.(J Card Surg 2002;17:502‐511)

Deferoxamine-fibrin accelerates angiogenesis in a rabbit model of peripheral ischemia

The intramuscular (IM) injection of a modified fibrin meshwork plus deferoxamine was tested in a rabbit model of acute hind-limb ischemia. After excision of the left external iliac and femoral arteries, 12 rabbits at the Milwaukee Heart Institute were divided into two groups: control and fibrin meshwork plus deferoxamine (FDEF) IM. The rabbits underwent angiography before surgery, immediately after, and 1 month postoperatively. These data were compiled through counting by means of a grid overlay. Another 12 rabbits at the Vakhidov Center of Surgery, which did not undergo angiography, underwent lower limb-calf blood pressure (LCBP) measurements made immediately after surgery and at postoperative days 10, 20 and 30. Biopsies from thigh skeletal muscles of rabbits that had L-CBP measurements underwent alkaline phosphatase staining on day 30 to determine the percentage of biopsied area that was occupied by capillaries. The number of arteries and arterioles crossing 71 grid intersections immediately post-surgery decreased from 30.2 6 2.3 to 18.0 6 2.0 (p, 0.05). One month post-surgery this number increased to 29.2 6 2.4 in controls (p, 0.05 vs immediately post-surgery) and to 59.6 6 3.2 in the FDEF group (p, 0.001 vs immediately post-surgery). By day 30 the L-CBP ratio improved in the FDEF group (0.8 6 0.02) vs controls (0.3 6 0.04). By day 30 the capillary density increased from that of normal muscle tissue (198.6 6 12.9/mm2) to 292 612.4/mm2 in the FDEF group (p, 0.05), but decreased in the control group to 98.7 6 7.7/mm2. IM injection of FDEF considerably accelerated angiogenesis in severely ischemic hind-limb tissue in this model, making it a viable treatment method for clinical use in patients who have critical limb ischemia.

Electrical stimulation promotes angiogenesis in a rabbit hind-limb ischemia model

In previous investigations, it was shown that applying a modest regimen of electrical stimulation (ES), even in severely ischemic tissue, improves the healing process, accelerates neovascularization, and enhances angiogenesis in muscle tissue. Our objective in this current report was to further understand ES as a potential alternative treatment for severe muscle ischemia. Immediately after the left distal external iliac artery and the femoral artery were excised, ES (30 contractions per minute [cpm], 2 V, single impulses per burst) was applied to rabbit adductor muscle near the site of the excised femoralis artery for 24 hours daily over 1 month. Three other series served as controls: ES without arterial excision; arterial excision without ES or lead implantation; and arterial excision with lead implantation but no ES. Histologic study of capillary density was performed by angiography (employing a grid template) and by measuring the lower limb-calf blood pressure ratio. At the end of 30 days in the ES series, 10.5 ±1.2 contrast-medium opacified arteries (COAs) crossed a specific grid section segment compared with 7.2 ± 1.5 in the control series without ES (p < 0.05); 68.2 ±9.3 COAs crossed a grid section compared with 43.2 +6.4 in controls (p< 0.05); 27.3 ± 1.2 grids contained COAs compared with 29.3 ±3.5 in controls (p<0.05); lower limb-calf blood pressure ratio was 0.81 ±0.06 compared with 0.31 ±0.07 in controls (p <0.05); and capillary density was 283.7 ±24.5 mm2 compared with 91.4 ±20.9 mm2 in controls (p <0.001). These preliminary results show that cautious ES enhances and accelerates muscle revascularization in severely ischemic tissue.

Comparison of Different Regimens of Electrical Stimulation Applied to Nonmobilized and Newly Mobilized Latissimus Dorsi Muscle

Abstract We investigated the possibility of preventing further aggravation of muscle ischemia and necrosis in newly mobilized, unconditioned latissimus dorsi muscle (LDM) by utilizing short increments of stimulation with intervening rest periods. Adult St. Croix sheep (N = 12) weighing 30 ± 8 kg were used in this study. Fatigue tests (30 min) using different stimulation regimens before and after LDM mobilization were performed on all animals; the length of time to return to baseline levels was also measured. Our investigation yielded results that contradict the conventional wisdom that any electrical stimulation damages newly mobilized LDM and will cause a considerable decrease in contractile force (CF). Stimulation regimens using continuous contractions at 30 and 60 contractions per minute (CPM) for 30 minutes were damaging to the LDM. CF also dropped significantly and returned slowly to baseline values: at 60 CPM, CF dropped to 50 ± 4% and did not return to baseline even after 90 minutes of rest; at 30 CPM, CF dropped to 61 ± 4% and baseline was restored after 80 minutes of rest. Electrical stimulation using continuous contractions at a slower rate (15 CPM) was tolerable, although a 23% decrease in CF was noted (p < 0.05 when compared to 60 CPM). These results did not satisfy us that such a regimen would be useful for cardiac assistance immediately after cardiomyoplasty. The work‐rest regimen at 30 CPM also gave poor results: CF decreased to 75 ± 2% and baseline was restored after 80 minutes of rest. Promising results were seen when utilizing a work‐rest regimen at 15 CPM. The newly mobilized LDM showed no visible signs of fatigue: CF decreased minimally to 92 ± 3% (p < 0.05 when compared to 30 CPM), and light microscopic analysis of biopsies revealed no morphological damage exceeding that typically seen after subtotal mobilization. Such results open avenues for future investigations: beginning electrical stimulation immediately after cardiomyoplasty (using a single impulse and a slow rate of contraction); decreasing the length of time necessary to obtain full cardiac assistance; and beginning partial cardiac assistance immediately after cardiomyoplasty (if needed) for approximately 30 minutes several times a day.

The use of biologic glue for better adhesions between the skeletal muscle flap and the myocardium and for increasing capillary ingrowth

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Is it possible to perform immediate cardiac assist using untrained latissimus dorsi muscle in a work-rest regimen?

The authors investigated what contractile force (CF) could be obtained from unconditioned latissimus dorsi muscle immediately after mobilization and for the 2 week vascular period of recovery. Latissimus dorsi muscle mobilization was performed on seven adult (4 experimental and 3 control) sheep leaving only the pedicle and the peripheral muscle intact. Telectronics stimulators (Myostim 7220; Teletronics Pacing Systems, Inc, Englewood, CO) were implanted. Immediately after mobilization 11-35% of the initial CF was lost. A 30 min fatigue test was performed 1 hr after mobilization (20 g/kg preload, 10 V, 10 Hz, 15 BPM, 6 impulses per burst) using a 1 min work-1 min rest regimen. Two sheep lost 2-12% of initial CF; two increased CF by 14-24%. At the end of the fatigue test, CF consisted of 74-89% of immobilized CF. Electrical stimulation training of the muscle was then initiated with the following regimen in the experimental animals only: 15 BPM, single impulses, 5 V, 10 Hz. Every day the muscle was exercised using a work-rest regimen to mimic cardiac assist, starting with 20 min on day 2, and increasing by 2 min per day until a total of 50 min was reached on day 16. All animals were retested for CF using a 42 min fatigue test on days 6, 11, and 16. On day 6, there was no fatigue evident in the experimental group during the 42 min test. CF after testing was 59-81% (mean 67%) of initial data. In the control group (animals with no electrical stimulation training protocol), CF decreased by 11% (from 64 to 53%). On day 11, there was no fatigue evident in the experimental group; CF in all animals increased by 2-8%. On day 16, there was also no fatigue evident in the experimental group; CF increased by 0-9%. An additional 20 min of continuous contraction (15 BPM) fatigue testing was performed on the muscle without rest between the tests. No fatigue was evident at the end of testing. Light microscopic analysis of latissimus dorsi muscle biopsy specimens taken on the days of testing showed no evidence of necrotic damage. Our investigations suggest that it may be possible to start muscle transformation immediately after mobilization and use the untrained latissimus dorsi muscle for cardiac assist immediately after surgery for short periods.

Age related skeletal muscle response to electrical stimulation.

We hypothesized that the conditioned muscles of elderly and growing organisms have different responses to electrical stimulation from that of young adult organisms. Five day old lambs, 1 year old sheep, and 8 year old elderly sheep were used for this investigation. The latissimus dorsi muscle (LDM) was partially mobilized and left in situ. Two electrodes were implanted and electrical stimulation (ES) was begun for 8 weeks; it was then stopped for 2 weeks. Biopsies were taken before ES, after 8 weeks of ES, and after the 2 week delay period. The LDM of old sheep has less fatigue resistance than the LDM of younger animals. Conditioned LDM of the lamb continued to be fatigue resistant after a 2 week delay compared with adult sheep. In all animals, lactate dehydrogenase (LDH) fraction five decreased and LDH-1 + 2 fractions increased after ES. After a 2 week delay, the data returned to baseline values only in adult animals. The percentage area occupied by mitochondria in old sheep was less after ES than in younger animals. In all animals, the mitochondrial area increased after ES and reverted to baseline values after the delay. The number of nuclei and fibers considerably increased after ES. Only in the lamb did the number of nuclei and fibers continue to be elevated after the delay. There are more changes in young skeletal muscle than in adult (1 year or 8 year old) muscle during ES, and they “remember” these properties. Elderly skeletal muscle does not convert to a fatigue resistant state as completely as adult skeletal muscle during a conventional 8 week ES protocol. It is necessary to change and prolong the ES protocol for elderly patients.