Cutaneous Maximus Research Articles

Mixed Metal Oxide Nanoparticle Formulations for the Treatment of Seroma

Seroma formation is a well-recognized postoperative complication for many plastic and general surgical procedures. Although various tissue adhesives and substances have been used in an effort to treat seroma formation, no therapies have been established clinically. Recently, the nano-bridging phenomenon has been introduced as a promising approach to achieve tissue adhesion and strong closure of deep skin wounds in rats. The present study seeks to assess the potential of nano-bridging beyond skin wounds in a rat model of seroma. Seromas were induced in 20 Lewis rats through bilateral axillary lymphadenectomy, excision of the latissimus dorsi and cutaneous maximus muscles, and disruption of dermal lymphatics. On postoperative day (POD) 7, the seroma was aspirated on both sides. A bioactive nanoparticle (NP) suspension based on zinc-doped strontium-substituted bioglass/ceria nanoparticles (NP group) or fibrin glue (fibrin group) was injected into the right seroma cavity, while the left side was left untreated. On POD 14, the NP group showed complete remission (no seromas at all), while the fibrin group recorded a reduction of only 63% in the seroma fluid volume. The NPs exerted local anti-inflammatory and neo-angiogenic effects, without any detectable systemic changes. Moreover, the ceria levels recorded in the organs did not surpass the background level, indicating that the nanoparticles stayed at the site of application. This study is a promising first example demonstrating the ability of inorganic nanoparticle formulations to reduce seroma formation in a rat model, without any detectable systemic adverse effects. These results emphasize the potential of nanotechnological solutions in the therapeutic management of seroma in the clinical setting.

Tissue-specific activities of the Fat1 cadherin cooperate to control neuromuscular morphogenesis.

Muscle morphogenesis is tightly coupled with that of motor neurons (MNs). Both MNs and muscle progenitors simultaneously explore the surrounding tissues while exchanging reciprocal signals to tune their behaviors. We previously identified the Fat1 cadherin as a regulator of muscle morphogenesis and showed that it is required in the myogenic lineage to control the polarity of progenitor migration. To expand our knowledge on how Fat1 exerts its tissue-morphogenesis regulator activity, we dissected its functions by tissue-specific genetic ablation. An emblematic example of muscle under such morphogenetic control is the cutaneous maximus (CM) muscle, a flat subcutaneous muscle in which progenitor migration is physically separated from the process of myogenic differentiation but tightly associated with elongating axons of its partner MNs. Here, we show that constitutive Fat1 disruption interferes with expansion and differentiation of the CM muscle, with its motor innervation and with specification of its associated MN pool. Fat1 is expressed in muscle progenitors, in associated mesenchymal cells, and in MN subsets, including the CM-innervating pool. We identify mesenchyme-derived connective tissue (CT) as a cell type in which Fat1 activity is required for the non–cell-autonomous control of CM muscle progenitor spreading, myogenic differentiation, motor innervation, and for motor pool specification. In parallel, Fat1 is required in MNs to promote their axonal growth and specification, indirectly influencing muscle progenitor progression. These results illustrate how Fat1 coordinates the coupling of muscular and neuronal morphogenesis by playing distinct but complementary actions in several cell types.

Length-dependent axo-terminal degeneration at the neuromuscular synapses of type II muscle in SOD1 mice

In motor neuron diseases, there is a prolonged period of time before any clinical symptoms begin to appear. During this time, distal axonal degeneration, or “dying back” axonopathy, begins to occur before the onset of clinical symptoms and motor neuron death. This preclinical degeneration is a hallmark of motor neuron diseases in both animal models and human patients. Generally, in muscles with mixed fiber types, distal degeneration occurs in fast-fatigable α-motor axons innervating type IIb muscle fibers before axons innervating slow, type I muscle fibers. We investigated whether the “dying back” axonopathy in a pure fast-fatigable α-motor axon nerve is a length-dependent process. The lateral thoracic nerve (LTN) exclusively consists of motor nerves that innervate the very thin cutaneous maximus muscle (CMM) that solely contains type II neuromuscular synapses. We characterized the LTN and CMM synapses both morphologically and physiologically in the superoxide dismutase 1 (SOD1) mutant mouse model of amyotrophic lateral sclerosis (ALS). By 60days of age, there was a significant “dying back” phenomenon at the caudal region while the rostral region remained intact. The longer axons innervating the caudal region appear to be more susceptible to degeneration in the SOD1 mouse indicating that the axonal degeneration of motor neurons innervating type II fibers is a length-dependent process. Additionally, we identified how the simplicity of the LTN–CMM system offers a better method to investigate axon degeneration in an ALS mouse model and may be used to investigate possible therapeutic compounds for axon protection and regeneration.

Lateral thoracic artery perforator-based flap: a new experimental model

BackgroundThe aim of this study was to describe a new experimental perforator-based flap in rats, supplied by lateral thoracic artery perforator. MethodsThrough out the study, two control and two experimental groups were created consisting of six rats in each group. In the first control group (group 1), mid-axillary line and the fourth intercostal space intersection were used as the center of the flap. A 3 × 2 cm flap was designed and elevated above the cutaneous maximus muscle by transecting all connections with the muscle. In the second control group (group 2), the flap was extended caudally, and 3 × 6 cm flap was designed. Then, the flap was elevated in the same manner as it was described for group 1. In the first experimental group (group 3) 3 × 2 cm flap and in the second experimental group (group 4) 3 × 6 cm flap were designed like control groups. The flaps were islanded on a single musculocutan perforator arising from lateral thoracic artery. The surviving skin paddle areas were calculated on postoperative day 7. ResultsThe flap viability was calculated 0% for control groups (groups 1 and 2), 100% for the first experimental group (group 3), between 33.3% and 37.7% for the second experimental group (group 4; mean ± standard deviation, 34.76% ± 1.92%). ConclusionsThis new lateral thoracic artery perforator-based flap has a constant anatomy and reliable survival pattern. Also, easy harvesting and the possibility of designing two flaps per animal make this new flap an appealing model for pathophysiological or pharmacologic researches.

Neo-vascularisation of musculocutaneous and muscle flaps after division of the major vascular supply: An experimental study

Experimental studies have shown that musculocutaneous flaps are not dependent on a major pedicle for survival after 7-8 days, as revascularisation occurs from surrounding well-nourished tissue. However, muscle component loss in myocutaneous flaps after pedicle division has been reported. No study that examines the nature of the vascular ingrowth from underlying beds by blocking the peripheral cutaneous beds has been conducted in musculocutaneous and skin-covered muscle flaps. This study was designed to investigate the origin of the dominant source of neo-vascularisation after interruption of the major vascular supply in island musculocutaneous and island skin-covered muscle flaps by blocking neo-vascularisation from wound edges and the recipient bed. Twenty-eight rats were divided into four experimental groups. In group I, a cutaneous maximus musculocutaneous island flap (MCIF) was raised, and the wound edges of the flap were blocked with a silastic sheet. In group II, an MCIF was raised, and the recipient bed was blocked with silastic. In group III, an island cutaneous maximus muscle flap (IMF) was raised as an island flap covered by a full-thickness skin graft (FTSG), and the wound margins were blocked with silastic. In group IV, an IMF was raised as an island flap covered by an FTSG, and the recipient bed was blocked with a silastic sheet. On the seventh postoperative day, vessel ligation was performed in each animal. Microangiographic studies and histopathological evaluations were performed 14 days after the first operation. In microangiographic studies, neo-vascularisation was more prominent in groups II and IV (the groups in which the recipient beds were blocked) than in groups I and III (the groups in which the wound edges were blocked). Upon histopathological examination, the number of vessels was significantly lower in group I and group III than in group II and group IV (p<0.001). Our findings revealed that neo-vascularisation from either the recipient bed or the wound edges was sufficient to ensure full flap survival in musculocutaneous flaps, and skin-grafted muscle flaps do not need major axial vessels 7 days after flap elevation in rats if the recipient bed or wound edges are well-vascularised. The results also indicated that revascularisation mainly comes from the peripheral wound edges and is independent of flap type.

The lateral thoracic nerve and the cutaneous maximus muscle—A novel in vivo model system for nerve degeneration and regeneration studies

We report a novel in vivo mouse model system to study regeneration of injured motor nerve and spatiotemporal pattern of denervation in experimental nerve diseases. The lateral thoracic nerve (LTN), as a pure motor nerve, innervates the cutaneous maximus muscle (CMM) by some of the shortest and the longest motor nerve fibers in the mouse body. Its branches and nerve terminals can be imaged in whole mount preparations. Here we describe the branching pattern of the LTN and its innervation of the CMM, and characterize degeneration and regeneration over time after a LTN crush by morphological and electrophysiological analyses. We demonstrate the utility of this model in a well-established neurotoxicity paradigm and in a genetic disease model of the peripheral neuropathy. Furthermore, this system enables punch biopsies that allow repeated and multi-location examinations for LTN regeneration and CMM reinnervation over time. The presence of the LTN and the CMM in a variety of species and its easy accessibility suggests that this in vivo model system offers considerable promise for future nerve degeneration and regeneration research.

Deal Breaker: Semaphorin and Specificity in the Spinal Stretch Reflex Circuit

Stretch reflex circuits are a prime example of wiring specificity in the vertebrate spinal cord. Homonymous sensory afferents and motoneurons typically form monosynaptic connections, while neurons innervating antagonistic or unrelated muscles do not. Pecho-Vrieseling et al. now show that the semaphorin Sema3E and its receptor Plexin-D1 prevent monosynaptic connectivity in the cutaneous maximus muscle stretch reflex circuit.

Cross-site anastomosis for a free flap with a long pedicle in the rat

We describe a simple and reliable technique for tunneling of the vascular pedicle of the free flap with a long pedicle, that may otherwise cause twisting and/or kinking of the pedicle. There are two modifications of the technique. In the cross-groin anastomosis modification, using a haemostat in a blunt manner, a suprapubic subcutaneous tunnel is created between two groin regions. The vascular pedicle of the flap is pulled and passed through this tunnel to the contralateral region. While the anastomoses are done on one site, the flap is inserted to the contralateral groin. In the second modification ('groin anastomosis-cross dorsum flap insert procedure'), the flap is transferred to the dorsum of the rat at approximately the same level of the groin. Using a haemostat, a large subcutaneous tunnel safe enough for the pedicle is created between the groin region and the rat dorsum lateral to the hip region. The anastomoses are done in the groin region, and the flap is inserted on the dorsum. This technique was used in 16 animals, with a free cutaneous maximus muscle musculocutaneous flap transfer based on the axillary vessels being done in eight animals; and a posterior thigh perforator-based flap based on the popliteal vascular pedicle in the remaining eight. Both groups had a pedicle length of approximately 4 cm, with a 100% survival rate. In conclusion, the technique is simple, reliable, and can be easily carried out without necessitating special procedures. It provides a straight course for the vascular pedicle with tension-free anastomosis by appropraitely adjusting the distance of the vascular anastomosis and flap insertion site.

The role of the ETS gene PEA3 in the development of motor and sensory neurons

The ETS family of transcription factors includes two members, ER81 and PEA3, which are expressed in groups of sensory and motor neurons supplying individual muscles. To investigate a possible role of these genes in determining sensory and/or motor neuron phenotype, we studied mice in which each of these genes was deleted. In contrast to the deletion of ER81, which blocks the formation of projections from muscle sensory neurons to motor neurons in the spinal cord, deletion of PEA3 causes no obvious effects on sensory neurons or on their synaptic connections with motor neurons. PEA3 does play a major role in the formation of some brachial motoneurons however. Motoneurons innervating the cutaneous maximus muscle, which are normally PEA3 +, fail to develop normally so that postnatally the muscle is innervated by few motoneurons and is severely atrophic. Other studies suggest that these motoneurons initially appear during development but fail to contact their normal muscle targets.

ICE REDUCES EDEMA

Ice is applied following a soft-tissue injury on the basis of clinical information. This study investigates the relationship between ice therapy (cryotherapy) and edema by determining microvascular permeability before and after contusion with and without ice therapy and provides data supporting a reduction in edema following cryotherapy. A dorsal microvascular chamber was created in rats to allow the direct examination of microvascular parameters in intact, pre-established microvascular beds of the cutaneous maximus muscle in conscious rats. The rats received a contusion or sham contusion and were treated with cryotherapy or were not treated. Microvascular permeability (edema) was assessed by measuring fluorescent-labeled albumin in the interstitial fluid before and after contusion. Microvascular permeability following contusion was significantly increased in the group that received the contusion without cryotherapy compared with that in the group that received the sham contusion without cryotherapy (control) (p < 0.001). When ice was applied fifteen minutes after the contusion for twenty minutes, microvascular permeability (edema) decreased significantly (p < 0.001) compared with that in the group that did not receive cryotherapy after contusion. Permeability was increased in the group that received cryotherapy following the contusion compared with that in the control group (p = 0.012), although the increase was not as great as that between the group that received the contusion without cryotherapy and the control group. Sham contusion with cryotherapy significantly reduced microvascular permeability compared with that in the control group (p = 0.004). Sham contusion without cryotherapy did not cause a significant change in the microvascular permeability of postcapillary venules after 300 minutes compared with baseline measurements. The application of ice significantly decreased microvascular permeability following striated muscle contusion. The results of this study demonstrated that microvascular permeability is increased following a contusion coincident with significant leukocyte-endothelial interactions. However, microvascular permeability was significantly reduced following cryotherapy, a treatment demonstrated to reduce the number of rolling and adherent leukocytes. This association suggests that the reduction in edema in injured skeletal muscle following cryotherapy may be due to a reduction in leukocyte-endothelial interactions.

MONITORING EARLY CHRONIC GRAFT VASCULOPATHY AFTER HEART TRANSPLANT THROUGH SIMULTANEOUSLY TRANS-PLANTED SKELETAL MUSCLE

424 Routine endomyocardial surveillance biopsy after cardiac transplantation carries significant risk and has poor sensitivity for diagnosing chronic graft rejection or early graft vasculopathy. We have previously shown close correlation in acute rejection findings in a rat model of simultaneous allotransplantation of heterotopic heart (HH) and cutaneous maximus muscle flap (MF). The aim of this study was to evaluate the degree of correlation of rejection patterns in a rat model of chronic rejection. METHODS: 13 Lewis rats (RT1′) underwent heterotopic heart (HH) and cutaneous maximus muscle flap (MF) allotransplants from Brown-Norway (RT1″) donors in three experimental groups: Group I (simultaneous HH and MF, n=6). Group II (HH alone, n=4) and Group III (MF alone, n=3). Allograft recipients were given cyclosporine for 10 days after transplant. Nine transplants were performed between Lewis rats and served as controls: simultaneous HH and MF (n=5). HH alone (n=3). and MF alone (n=2). Grafts were evaluated 83-93 days post-operatively by standard and immunohistochemical studies. The grade of rejection was assessed using ISHLT criteria. RESULTS: At 3 months post-transplant, Group I had 6/6 HH and 5/6 MF viable grafts. One MF was lost due to ischemic necrosis. Findings diagnostic of acute cellular rejection (Grade IIIA/IIIB) were noticed in 5/6 HH grafts and in all 5 viable MF grafts. Chronic vascular changes consistent with early graft vasculopathy and chronic rejection were present in 6/6 HH grafts and in the 5 viable MF grafts. Groups II and III examined isolated HH or MF grafts and showed similar patterns of chronic vascular rejection, vasculopathy and cellular rejection as in Group I. Rejection was identified as 'moderate' in 5 of 7 single and 5 of 6 simultaneous allografts, which showed no change in rejection severity associated with additional transplanted tissue (p=0.61). As expected, control group grafts showed no evidence of rejection, with minimal muscle atrophy. CONCLUSIONS: This rat model of simultaneous allotransplantation of HH and MF provides a method of evaluating chronic rejection. We observed a close correlation in severity of vascular changes between simultaneously transplanted HH and MF grafts. The rejection severity seen in HH and MF allografts is similar whether transplanted alone or together which may indicate the relative immunologic safety of simultaneous grafting. This new technique of evaluating vascular rejection and early graft vasculopathy may be useful in monitoring cardiac allografts for chronic rejection.

Salvage of free flaps after secondary venous ischemia by local delivery of heparin.

Recent studies have demonstrated that heparin may protect against reperfusion injury through a direct effect on the microvascular endothelium that is independent of its effect on systemic coagulation. The purpose of this study was to determine whether local delivery of low-dose heparin has a role in the salvage of musculocutaneous flaps after secondary venous ischemia and revascularization. Cutaneous maximus musculocutaneous flaps were transplanted to the contralateral groin in adult Sprague-Dawley rats. All flaps were subjected to 2 hours of primary arteriovenous ischemia followed by 20 hours of reperfusion. The flaps were then subjected to a 6-hour secondary venous ischemic insult followed by anastomotic revision and reperfusion. Animals in group I received no adjunctive treatment. Those in group II were treated with low-dose heparin (5-6 U per kilogram per hour) infused systemically via the inferior epigastric vein. Those in group III received the same dose of heparin infused locally into the flap via the inferior epigastric artery. The dose of heparin used in groups II and III was insufficient to prolong the activated partial thromboplastin time above normal values. At 7 days, mean flap necrosis was 60.8% in group I and 62.1 in group II. Local heparin delivery (group III) resulted in complete survival of all flaps. Histological examination after 48 hours of reperfusion demonstrated improved microvascular patency and reduced neutrophilic infiltration in the flaps of group III animals. Thus, local infusion of low-dose heparin resulted in significantly improved flap salvage through a mechanism independent of its effect on systemic coagulation.

REJECTION PATTERN OF SIMULTANEOUSLY TRANSPLANTED CARDIAC AND SKELETAL MUSCLE IN A RAT MODEL.

408 Purpose: We sought to correlate cardiac and skeletal muscle allograft rejection in a heterotopic heart transplant rat model for the following purposes: 1) to quantitate the degree and severity of skeletal muscle rejection based on the grading system for cardiac muscle rejection, and 2) to assess the feasibility of use of biopsies from simultaneously transplanted skeletal muscle for surveillance of cardiac rejection.Methods: 34 Lewis (RT11) rats underwent simultaneous heterotopic cardiac and cutaneous maximus skeletal muscle transplants. Of those, 7 rats (Group I) received syngenic grafts from Lewis donors with no immunosuppression and served as control. The remaining 25 rats received allografts from Brown Norway (RT1n) donors in two groups: Group II(n=22) received no immunosuppression, and Group III (n=5) received one week of cyclosporine. Non-immunosuppressed rats (Groups I & II) were sacrificed at days 1 through 7 after the transplant whereas immunosuppressed rats (Group III) were sacrificed at 4 weeks. Tissue sections from the grafts were graded for cellular rejection based on the grading system of the Heart Rejection Study Group (International Society for Heart and Lung Transplantation, ISHLT).Results: All group I and III grafts were functional at 7 days post-transplant with no evidence of rejection. Of group II, 2 rats showed no signs of cardiac or skeletal muscle rejection when sacrificed on day 1. All the other rats (n=20) developed graft rejection within one week after the transplant. Cellular rejection correlated grade for grade in 17/20 (85%). Of the three rats that did not show grade to grade correlation, the cardiac biopsy was interpreted as Grade IA in one case when the skeletal muscle showed no rejection. In each of the other two cases, the heart showed focal solitary lymphocytic infiltrate with myocyte replacement (Grade II), and the muscle flap showed diffuse perivascular and interstitial lymphocytic infiltrates without myocyte necrosis (Grade IB). The severity of rejection correlated in 19/20 (95%) rats: mild rejection (Grades IA, IB, II): 9/10 rats (90%); moderate (IIIA, IIIB): 6/6 (100%) and severe (IV): 4/4 (100%).Conclusion: Skeletal muscle rejection may be quantitated using the ISHLT grading system for cardiac muscle rejection. Cardiac and skeletal muscle allografts had similar pattern and severity of rejection with little grade to grade variability. The clinical implications of this correlation for surveillance of cardiac rejection warrants further investigation.

A new rat model to study the correlation of cardiac and skeletal muscle allograft rejection.

As a first step to study the correlation of cardiac and skeletal muscle allograft rejection, we describe a new experimental rat model of simultaneous heterotopic heart and cutaneous maximus muscle flap allotransplant. Brown Norway rats were used as donors and Lewis rats as recipients. No immunosuppression was given. The grafts were revascularized with sequential end-to-side anastomosis of each vascular pedicle to the infrarenal aorta and vena cava. Syngeneic heart and cutaneous maximus muscle grafts remained functional and showed no sign of rejection at 7 days after the transplant. In contrast, both allografts developed severe rejection and functional compromise at 7 days after the transplant. Our experimental model is technically feasible and reproducible and may provide important information about the pattern of rejection of cardiac and skeletal muscle allografts.

Manual of Experimental Muscle Flap and Organ Transplantation Models in the Rat

The Manual of Experimental Muscle Flap and Organ Transplantation Models in the Rat by Zhang and co-authors represent an impressive compilation of detailed and valuable anatomical and surgical information for laboratory investigators employing microsurgical rat models. Certainly, this well-organized and well-written manual will be a valuable addition to the reference shelf in all laboratories employing rats as surgical models of human disease. The manual is organized into three sections. The first of these concerns basic information about rat handling, anesthesia, surgery, as well as general information about the utility of animal models in basic research. This section will serve as a good introduction for the novice rat surgeon. The second section is entitled, “Muscle and musculocutaneous flaps,” but is in fact the description of virtually every free tissue transfer (except organ transplants) that has been described in the rat. There are separate chapters on the serratus anterior, latissimus dorsi, rectus abdominus, gracilis, gastrocnemius, cutaneous maximus, and pectoralis major muscle and, if applicable, myocutaneous flaps. In addition, the groin free flap, the temporoparietal fascial flap, and the free toe transfer are covered. In each chapter, a rational well-organized discussion of the general utility of the flap in experimental surgery, of the relevant anatomy for each flap, and of the surgical technique, is provided. The detailed, step by step descriptions of the surgical procedures for elevation and transfer of these flaps are especially useful. The author’s vast experience with these techniques is evident in the numerous, important subtle technical points which are emphasized. In section three, the use of microsurgical models of whole organ transplantation is covered. An introductory chapter points out some of the history of organ transplantation in animal models and discusses some important aspects of experimental transplantation surgery, including immunosuppressants. After a chapter on hindlimb transplantation, the remaining chapters cover bowel, kidney, liver, pancreaticoduodenal, heart, testicular, and ovarian transplantation. These chapters provide the same organized and detailed description of the operations as in section two. In all of the chapters in sections two and three, welldrawn, accurate illustrations provide an invaluable resource for the relevant anatomy and the surgical execution of each of the procedures described. These illustrations alone would make this manual a worthwhile addition to any microsurgical laboratory. Like all publications, this manual does have several deficiencies, which the authors could perhaps address in a subsequent edition. Although the description of animal handling is quite useful, no mention is made of the importance of strict adherence to the National Institutes of Health Guide for the Use and Care of Laboratory Animals (NIH Publication 86-23). Secondly, this multi-author manual has chapters by a number of people who are not listed in the list of authors on the cover. It would probably be most appropriate to indicate that Zhang, Lineaweaver, Kao, Walker, and Tonken have in fact edited this multi-author manual, and then to acknowledge each of the authors in a separate location. In addition, this manual has been obviously published with the aid of commercial sponsorship. Although the name of the company appears prominently on the front and

Microvascular responses to body tilt in cutaneous maximus muscle of conscious rats

We investigated microvascular responses to head-up tilt (HUT) and head-down tilt (HDT) in striated muscle of conscious male rats (n = 15; body wt 163 +/- 5 g). To observe the microcirculation in the cutaneous maximus muscle, a transparent polycarbonate chamber (1.5 cm diam) was implanted aseptically into a skin fold created between the shoulders. Rats were trained to sit quietly during HUT and HDT while positioned on a horizontal microscope that rotated in the sagittal plane. At 4-5 days after surgery, arteriole and venule diameters were recorded using videomicroscopy while the rat experienced 10 min each (in random order) of HUT or HDT at 20 degrees or 40 degrees separated by 2-h rest periods. HUT had no affect on microvessel diameter; 20 degrees HDT had little affect. In response to 40 degrees HDT, "large" arterioles (88 +/- 18 microns; n = 10) constricted by 18 +/- 2% (P < 0.05) and "small" arterioles (40 +/- 17 microns; n = 7) dilated by 21 +/- 3% (P < 0.05); this difference suggests variation in mechanisms controlling arteriolar responses. Venules (45 +/- 22 microns; n = 9) exhibited a larger fluctuation in diameter (amplitude 13 +/- 5 microns) during 40 degrees HDT compared with other body positions (amplitude typically 2-3 microns), suggesting that venomotor activity may be induced with sufficient fluid shift or change in central venous pressure. These observations illustrate a viable model for studying microvascular responses to gravitational stress in conscious rats.

Effect of Age upon Ischemia/Reperfusion Injury in Rat Muscle Free Flaps

Metabolic and functional changes have been found in tissues of aging man and animals. It is not known if old age has a detrimental effect on the outcome of free tissue transfer or extremity replantation, nor has it been determined if prolonged ischemia may exacerbate such effects. To explore these issues, we utilized a syngeneic rat model of cutaneous maximus muscle transplantation, isolating the effects of flap age and ischemia on flap survival and metabolic function. Flaps were raised in young adult (2-3 months), middle-aged (10-12 months), and old (20-22 months) Lewis rats and transplanted to young Lewis recipients after 1, 6, or 10 hr of room temperature ischemia. Reperfusion periods of 2 hr, 2 days, or 2 weeks were allowed, and flaps were harvested for histologic and histochemical evaluation. Flap weights significantly increased after reperfusion following longer vs shorter ischemia and in the old flaps versus young flaps (for 1 hr of ischemia) ( P < 0.05). Histology confirmed a greater extent of interstitial edema in flaps from older rats. Histochemical assessment of muscle dehydrogenase activity (nitroblue tetrazolium staining) demonstrated reduced staining in both the young 10-hr ischemic flaps and in the older 6 and 10-hr ischemic flaps. These results indicate that muscle does not tolerate ischemia as well in older animals, but that a short ischemic interval (1 hr) is well-tolerated.

Ex vivo perfusion with anticoagulated blood decreases ischemia/reperfusion injury

Rat muscle flaps (cutaneous maximus) were subjected to 4 hours of room-temperature ischemia followed by 44 hours of cold (4°C) ischemia before transplantation. Experimental flaps underwent a 15-minute ex vivo perfusion with heparinized-citrated blood between the warm and cold ischemic treatments; control flaps were not perfused. After 1 or 48 hours of recirculation, muscle dehydrogenase activity was found to be higher in the perfused flaps, indicating better muscle viability. Lipid peroxidation was lower in the perfused flaps, indicating a reduction in free radical generation. Histologic assessment revealed that perfused flaps underwent less injury than unperfused flaps. The findings of this study indicate that replacement of standing blood with anticoagulated blood, with the use of a brief ex vivo perfusion protocol, has a significant protective effect against ischemic injury. The experimental design mimics a realistic point of intervention and presents a paradigm for the clinical treatment of amputated extremities.

The influence of diabetes on free flap transfer: II. The effect of ischemia on flap survival.

Insulin-dependent diabetes mellitus causes microangiopathic changes in many tissues, including skin and muscle. It is not known if such changes are detrimental to free flap transfer, particularly after extended ischemia. To address this issue, we used an experimental design by using a syngeneic rat strain (Lewis) for free groin flap and muscle flap transplantations from streptozotocin-induced diabetic rats (2 month's duration of symptoms) to normal rats. Flaps from age-matched normal donors were transplanted to normal recipients for control comparisons. Groin flaps were stored ischemically for 12 or 18 hours at room temperature, or for 48 hours in the cold (4 degrees C) before transplantation. Flap survival and vascular patency were assessed at 7 days. Cutaneous maximus muscle flaps were transplanted to the groins of recipients after 6 hours of room temperature ischemia. Vascular patency, muscle viability, flap weight change (edema), and dehydrogenase activity were assessed after 2 days of reperfusion. Seventy percent, 67%, and 73% of diabetic groin flaps survived after 12, 18, or 48 (cold) hours of ischemia, respectively, in comparison with 90%, 73%, and 87% of normal flaps undergoing the same respective ischemia periods. The differences were not significant, even when the data were pooled (p greater than 0.1). Muscle flaps also showed no significant differences for the parameters studied. These results support the use of microvascular reconstructive surgery in diabetic patients, suggesting that moderate ischemic challenges do not compromise free flap transfer or extremity replantation.

Free flap transfer of the cutaneous maximus muscle in the rat: Comparison to the latissimus dorsi muscle flap

A new rat model of free muscle flap transfer is presented. Microvascular transplantation of the cutaneous maximus muscle flap is performed at the groin site, with anastomosis of the axillary vessels to the appropriate femoral vessels. This muscle flap has many useful attributes for experimental manipulation. It has a high success rate following transplantation, the anatomy is consistent, the dissection is straightforward, the length of pedicle is relatively long (10 mm), the vessels for repair are of sufficient size (1.0-1.35 mm diameter), and the microsurgical procedure can be performed in a relatively short period. The donor site deficit causes minimal impairment to animal mobility, and no evidence of limb ischemia is noted after ligation of the axillary vessels. The cutaneous area adjacent to the muscle is perfused by muscular perforators supplied by the flap pedicle; thus a skin island may be used to monitor the flap or to create a composite myocutaneous transfer. The cutaneous maximus muscle has mixed muscle types and anatomic dimensions similar to those of the latissimus dorsi muscle, and it provides ample tissue for pharmacological and biochemical studies, yet it presents easier dissection and microanastomoses than the latissimus flap, with more potential for versatility in application. The advantages of this muscle flap make it a very useful experimental model for flap transfer research.