Standard Cremaster Muscle Model for Ischemia Reperfusion

, Ilker Yazici2 and Maria Z. Siemionow3



(1)
Department of Vascular Surgery, Eskulap Hospital, Osielsko, Poland

(2)
Department of Plastic Reconstructive and Aesthetic Surgery, Istanbul Bilim University Faculty Of Medicine – Sisli Florence Nightingale Hospital, Istanbul, Turkey

(3)
Department of Orthopaedics, University of Illinois at Chicago, Chicago, IL, USA

 



Abstract

Ischemia – reperfusion injury (IRI) is a serious complication of revascularisation that biases a result of many surgical procedures. The pathophysiology of IRI is very complex and still not fully understood. For years a laboratory model of muscle ischemia reperfusion have been developed to provide reliable scientific tool for observation of IRI and investigation of possible therapeutic strategies. Cremaster muscle model of IRI plays an important role in research of IRI due to its similarity to clinical scenarios of limb ischemia, transplantation, replantation. This model provides unique opportunity to visualize important events that occur during reperfusion like capillary perfusion, leukocyte-endothelial interaction, microvasculature response and endothelial permeability. It is a potent model for intravital investigation of muscle microcirculation subjected to IRI.


Keywords
IschemiaReperfusionInjuryCremaster muscle flapModel


Abbreviations


EIA

External iliac artery

EIV

External iliac vein

FA

Femoral artery

FCD

Functional capillary density

FV

Femoral vein

IL

Inguinal ligament

IRI

Ischemia reperfusion injury

MPI

Microvascular permeability index

PPV

Preselected postcapillary venule



Introduction


In recent years we have witnessed a major development in medical sciences. We perform more and more complicated surgical procedures that require temporary ischemia. This complication arises following stroke, myocardial infarction, acute limb ischemia, mesenteric embolism, or organ replantation and transplantation [1, 2]. Provided that the ischemia has not been too prolonged, restoration of blood flow can salvage hypoperfused tissue. However, tissue damage can actually be increased during reperfusion by a group of pathogenetic mechanisms widely known as ischemia-reperfusion injury (IRI). This interesting process is triggered by commencement of blood circulation. In unfavorable conditions despite restoration of blood flow we observe no-reflow phenomenon. IRI complicates fate of transplanted and replanted limbs. It is a serious problem in acute limb ischemia where not so rarely it leads to limb loss or to whole organism complication like singly or multiple organ failure or even death.

Pathophysiology of ischemia reperfusion injury is very complex and not fully understood. Many hypotheses of vicious circle of IRI have been discussed for recent years. Well documented events during IRI is leukocyte attachment to endothelial cells, leukocyte migration to perivascular space where they release oxygen radicals and enzymes [3, 4]. Another events occurring during IRI is intravascular fibrin deposition and extravascular compartment edema [5]. One of key events in IRI cascade is translocation of phosphatidylserine in anoxic conditions to the surface of endothelial cells providing an attachment site for leukocytes and platelets [6]. This attachment impedes blood flow through the microvasculature and results in leukocyte activation and degranulation.

One of important research methods in understanding IRI is direct in-vivo observation of microcirculatory hemodynamics, vascular pathophysiology and leukocyte-endothelial interactions. Among many in-vivo observation methods a model of cremaster muscle is unique in field of IRI [710].

The first to introduce the cremaster muscle model in histological and electron microscopy studies on inflammation were Majno and Palade [7]. This model modified by Grant for use in microcirculatory observation by in vivo microscopy [8]. Baez was the first to open the cremaster muscle and to detach the testicle from the flap to spread out the flap and transilluminate it under a microscope [9]. The next step in improvement of the model was a tissue bath to provide better control of the microcirculatory environment introduced Miller and Wiegman [10]. This model was further developed by Siemionow in such a way that the cremaster muscle was completely isolated on its neurovascular pedicle, as an island flap [11]. As a result of this modification flap circulation is solely dependent on one feeding artery and one outflowing vein, excluding all other sources of vascular supply. This model simulates the conditions encountered during clinical surgery, where the main vasculature is interrupted resulting in total downstream tissue ischemia. These microcirculatory observations of the muscle flap made during ischemia and reperfusion expands significantly our ability to study microcirculation in relation to flap hemodynamics, reactivity of the microvessels, leukocyte-endothelial interactions and endothelial permeability [1, 11, 12].

Major advantage of the cremaster muscle flap is structural and functional similarity to other skeletal muscles. Thus, all changes observed in the microcirculation of this small flap could be applied to limb replantation or transplantation, muscle flaps used in clinical microsurgery as well as to vascular surgery. It allows observation and quantitative measurements of vasospasm, vessel diameters, formation of thrombus or emboli, capillary perfusion, leukocytes – endothelial interaction and endothelial damage. Interesting and valuable modification is a cremaster chamber model for chronic observation of the microcirculation and it’s response to IRI [12]. This model has been used to describe relation of thrombus formation at the anastomosis site of feeding vessels with decrease in capillary perfusion. To ease preparation of the model and avoid accessory parameters related with anastomosis, cremaster muscle flap is also used for vascular pedicle clamp related IRI [13]. In this setting flap is subjected to microcirculatory observation before vascular pedicle is clamped and during reperfusion 2–6 h later [14, 15]. This model may be also used for non-acute IRI in states of reduced blood flow. To induce reduced blood flow ligature is put on one or both of main flap vessels. Rat cremaster flap is also applicable to test local and remote pre or post ischemic conditioning.

For recent years many pharmacological compounds of different mechanism of action like, free radicals scavengers, antithrombotic agents, L-E adhesion inhibitors, immunosuppressive drugs and many other have been tested to alleviate IRI [1319].


Surgical Technique of Cremaster Flap Dissection


Male rats weighing about 150 g are used, because at this weight range the cremaster has optimal thickness, below 200 μm, enabling clear vision of microcirculatory bed of the flap. Another important feature is that the supplying vessels are large enough to allow reliable dissection of the vascular pedicle and ligation of all collateral branches.

Anesthesia is administered by subcutaneous injection of 6 mg/kg xylazine, 1 mg/kg acepromazine and 30 mg/kg ketamine in 0.3 ml. During observation animals are supplemented with s.q. injection of 3 ml of normal saline, and core body temperature was maintained at 36 ± 1 °C. Anesthesia is readministered at 1 h intervals in dose depending on the weight of the animal. The groin area is shaved and disinfected, the rat is prepped and placed in a supine position.

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Apr 2, 2016 | Posted by in Reconstructive surgery | Comments Off on Standard Cremaster Muscle Model for Ischemia Reperfusion
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