Michael J. Hickey

Prefabrication of thin transferable axial-pattern skin flaps: an experimental study in rabbits

The arteriovenous pedicle of all known axial-pattern skin flaps enters from the deep aspect and consequently the flap must contain fat and/or muscle and be of considerable thickness. In an attempt to fabricate a thinner axial-pattern flap the femoral artery and vein of rabbits were implanted, in various vascular configurations, directly into the subdermal layer of the skin. Implantation was found to provoke an extensive outgrowth of new vessels from the implanted artery and vein, and the progress and pattern of this neovascularisation was studied by carbon gelatine perfusion and histology. Neovascularisation begins within a few days of implantation and progresses rapidly. By 8 to 12 weeks it is possible to elevate regularly a viable, large skin flap based on the implanted pedicle.

Role of inducible nitric oxide synthase in the regulation of leucocyte recruitment

Constitutively produced nitric oxide released by endothelial cells has been shown to act as an endogenous agent which inhibits the rolling and adhesion of leucocytes in the microcirculation. However, during various types of inflammation, expression of the inducible form of nitric oxide synthase (iNOS) can dramatically increase the amount of nitric oxide present in tissues. Furthermore, as iNOS can be expressed by a wide variety of cell types, the distribution of nitric oxide is likely to be altered relative to that in unstimulated tissue. Under these conditions, it is less well understood whether iNOS-derived nitric oxide retains the anti-adhesive capabilities of constitutively produced nitric oxide. This review summarizes work done to examine this issue. Three main approaches have been used. In vitro studies have examined the role of iNOS in adhesive interactions between stimulated endothelial cells and leucocytes, providing evidence of an anti-adhesive effect of iNOS. In addition, the role of iNOS has been examined in vivo in animal models of inflammation using pharmacological iNOS inhibitors. These experiments were extended by the advent of the iNOS-deficient (iNOS(-/-)) mouse. Intravital microscopy studies of these mice have indicated that, under conditions of low-dose endotoxaemia, iNOS-derived nitric oxide can inhibit leucocyte rolling and adhesion. The potential mechanisms for these effects are discussed. In contrast, several other studies have observed either no effect or an enhancing effect of iNOS on inflammatory leucocyte recruitment. Taken together, these studies suggest that the importance of iNOS in modulating leucocyte recruitment can vary according to the type of inflammatory response.

The response of the rabbit rectus femoris muscle to ischemia and reperfusion

The rectus femoris muscle of the rabbit is perfused by a single artery and vein and is a valuable new model for study of ischemia-reperfusion injury of skeletal muscle. The consequences of increasing duration of ischemia to the rectus femoris have been examined. Postischemic muscle survival (means +/- SEM), as measured by Nitro blue tetrazolium (NBT) staining 24 hr after ischemia, was 90.5 +/- 1.5% after 2 hr normothermic ischemia, 77.1 +/- 7.7% after 3 hr, 41.8 +/- 7.6% after 3 1/2 hr, and 10.7 +/- 8.7% after 4 hr. Histology confirmed the NBT findings at 24 hr and showed considerable regeneration of muscle fibers 1-2 weeks after injury. The injury caused by 3 1/2 hr normothermic ischemia is the most suitable baseline for study of the effects of pharmacological agents in ischemic muscle injury. Further study of the effects of 3 1/2 hr ischemia by a quantitative Evans blue method revealed a rapid increase in vascular permeability commencing at the start of reperfusion and lasting for 5-6 hr. Vascular labeling with saccharated ferric oxide showed widespread labeling of venules within the injured muscle and electron microscopic examination showed severe injury to both leaking and nonleaking small blood vessels. However, increased vascular permeability accounted for only a small part of the increase in weight of ischemic muscle.

Microvascular submandibular gland transfer for the management of xerophthalmia; an experimental study

Free submandibular salivary gland transfer was investigated as a method of treatment for xerophthalmia. Xerophthalmia was created in rabbits by surgical removal of the lacrimal gland and the incidence of corneal ulceration was seen to correlate with the decrease in tear secretion. Microvascular transfer of the submandibular salivary gland to drain saliva into the conjunctival fornix prevented the development of corneal ulceration in 63% of the cases.

Microsurgical transfer of the greater omentum in the treatment of canine obstructive lymphoedema

In 13 dogs, experimental obstructive lymphoedema of the lower limb was created by combined radiotherapy and radical groin dissection. Six months later, when the degree of lymphoedema was stable, the lymphatic obstruction was bridged by microvascular insertion of a free omental graft. Six months after grafting, circumferential measurements indicated a statistically significant 38% reduction in the magnitude of lymphoedema. Biopsies showed the omental grafts were alive but contained much fibrous tissue. Lymphatic vessels were identified in 10 of 11 biopsies but connections between these lymphatics and lymphatics proximal to the graft could not be demonstrated by either lymphangiography or dye-injection techniques. The findings indicate that experimental obstructive lymphoedema in the dog can be reduced significantly by insertion of a vascularised omental graft. However, it could not be established that improvement was due to union of graft lymphatics with those of the lymphoedematous limb, although this union may have consisted of lymphatics too small to be demonstrated.

Mode of vascularization of control and basic fibroblast growth factor-stimulated prefabricated skin flaps.

&NA; This study, using 62 rabbits, examines the rate and pattern of vascular outgrowth from a subcutaneously implanted vascular pedicle, how the newly formed vessels connect to preexisting skin vessels, and whether local application of basic fibroblast growth factor can accelerate the angiogenic process. When the femoral artery and vein of rabbits are implanted beneath the skin, angiogenesis from both the pedicle and small blood vessels within the adjacent skin begins within 3 days. Perfusion with India ink reveals connections between the pedicle and dermal vessels as early as 5 days after implantation of the pedicle. Provided the pedicle does not thrombose, skin flaps based on it may survive completely when elevated as early as 2 weeks after implantation. Flap survival depends on the development of a small number of vascular connections between vessels arising from the pedicle and preexisting dermal vessels. If elevation is delayed until 4 weeks after implantation a flap may survive even if its pedicle has thrombosed. Prolonged release of basic fibroblast growth factor adjacent to the pedicle significantly increases the survival of flaps elevated 1 week after implantation but does not alter the survival of flaps elevated at 2 and 4 weeks. (Plast. Reconstr. Surg. 101: 1296, 1998.)

A morphological study of the long-term repair process in experimentally stretched but unruptured arteries and veins

As many avulsion amputations are incomplete and the vessels remain intact, the immediate pathology and long-term repair process (to 3 months post-injury) of experimentally stretched but unruptured rabbit femoral arteries and veins were examined. In stretched arteries, circumferential skip lesions involving endothelium, internal elastic lamina (IEL) and media occurred frequently and often up to 3 cm from the point of stretch. Medial smooth muscle cells (SMC) were significantly damaged or lost at lesions. Macrophages and neutrophils were found in lesions 1-4 days post-injury. Between 2-4 days, lesions were covered by endothelium and synthetic state SMC appeared in the media. At 1 week, a thin neointima (which persisted to 3 months) covered many lesions. The media at lesions gradually filled with SMC but generally remained disorganised even at 3 months post-injury. Stretching caused tears in vein walls, particularly close to the point of injury. There was no evidence of venous damage or repair in specimens examined 3 weeks and 3 months post-injury.

Effects of the endothelin receptor antagonist Bosentan on ischaemia/reperfusion injury in rat skeletal muscle.

We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 degrees C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17+/-2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48+/-7%) was significantly increased compared to the control group (P<0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles (P<0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.

Long term results of submandibular gland transfer for the management of xerophthalmia

Free submandibular gland transfer is potentially the best surgical solution to total xerophthalmia. Experimentally, gland function has only previously been followed for 2 months. This study was designed to investigate the long term effects of free transfer on gland function. Simultaneous creation of experimental xerophthalmia and free microvascular transfer of the submandibular gland to the orbit was completed in 10 rabbits. Two died before the end of the experimental period and the study was based on the remaining eight. Six months after transfer, five of the eight transferred glands were histologically normal and contained healthy neurones and nerve terminals. In these animals, tear secretion in the transfer eye was significantly greater than that of the control eye at 2 (p less than 0.05) and 6 months (p less than 0.02) after transfer. In the transfer eye, tear secretion was also found to have significantly increased from preoperative levels at 2 (p less than 0.05) and 6 months (p less than 0.01). The observed innervation of the transferred glands may play a role in maintaining secretion in the long term.

Effects of low dose intra-arterial monoclonal antibodies to ICAM-1 and CD11/CD18 on local and systemic consequences of ischaemia-reperfusion injury in skeletal muscle

The aim of this study was to investigate the effects of intra-arterial infusion of low doses of monoclonal antibodies (Mabs) against adhesion molecules (the neutrophil CD18 integrins, and the endothelial adhesion molecule, ICAM-1) on reperfusion injury in skeletal muscle. The rabbit rectus femoris muscle was rendered ischaemic for 2 1/2 hours. Mabs were infused (approximately 0.5 mg/kg) commencing 20 minutes before the end of ischaemia and for the first hour of reperfusion. 24 hours after reperfusion, the muscle was assessed for viability, oedema and neutrophil infiltration (myeloperoxidase (MPO) levels). The results of the viability assessment (control--20.9 (0-47.5)% [median (range)], anti-CD18--30.5 (3.0-89.4)%, anti-ICAM-1--27.9 (7.8-78.1)% and anti-CD18 combined with anti-ICAM-1--45.2 (15.6-92.3)%) showed no significant differences between groups, while analysis of MPO in the postischaemic muscle showed that the anti-ICAM-1 Mab reduced neutrophil infiltration significantly. Furthermore, in contralateral unoperated muscles MPO levels were elevated 24 hours after ischaemia in the contralateral muscle. This increased neutrophil infiltration was prevented by pretreatment with anti-ICAM-1. These results suggest that low doses of anti-CD18 and anti-ICAM-1 Mabs do not reduce reperfusion injury in skeletal muscle but may help to protect against systemic effects of severe trauma. The evidence suggests that reperfusion injury in this skeletal muscle model may be largely independent of neutrophil involvement.