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Do superficial epigastric veins of rats have valves?
BritishJournalofPlasticSurgery(2001),54, 151-153 9 2001TheBritishAssociationofPlasticSurgeons doi:10.1054/bjps.2000.3477 PLASTIC
SURGERY
Do superficial epigastric veins of rats have valves? L. Valdatta, T. Congiu*, A. Thione, M. Buoro, A. Faga and C. Dall'Orbo*
Plastic Surgery Section, and *Institute of Anatomy, University of lnsubria, Varese, Italy SUMMARY. The rat is a good model for experimental studies on the haemodynamic patterns of free-flap transfer but models involving the superficial epigastric vein have not been supported by the demonstration of the anatomical presence or absence of valves inside it. We performed a morphological study to assess whether or not valves are present inside the superficial epigastric vein of the rat. Segments of superficial epigastric veins were explanted from fifteen Sprague-Dawley rats. The lumen was studied with scanning electron microscopy after longitudinal or transverse incision of the venous walls. No valves were detected and we saw no evidence of their accidental removal. As most human veins do have valves, the clinical implications of haemodynamic data on reverse-flow island flaps obtained in rats must be considered very carefully. 9 2001 The British Association of Plastic Surgeons
Keywords: microsurgery, rat, venous valves.
The rat is a useful model for experimental microsurgery; its small size, resistance to infection and limited cost make it very suitable for experimental studies concerning the haemodynamic patterns of free flaps. Haemodynamic models involving the superficial epigastric bundle have been described, 1-3 focusing attention on the vascular anatomy 4 without specifying the presence or absence of valves inside the superficial epigastric vein. The presence of valves could be inferred from the results of experimental studies concerning the perfusion of the arterialised epigastric venous flap in the rat but, as far as we know, no study has clearly demonstrated the anatomic presence of venous valves inside the superficial epigastric venous channel. 5 We performed a morphologic study on the superficial epigastric vein of the rat to investigate the presence or absence of valves. Implications of the data collected for the venous outflow of experimental reverse-flow island flaps in rats are discussed.
I i
SUPERFICIAL EPtr~ TRUNK
Figure 1---The classical inguinal incision (continuous line) and our modifiedapproachthrough a postero-medialthigh incision (dashedline). arterial and nervous counterparts from its origin at the femoral vein to the bifurcation into its medial and lateral branches. The explanted specimens were divided into three groups in the early stages of our investigation:
Materials and methods We performed a study on 15 male Sprague-Dawley rats, weighing 350-400g, caged and fed as usual, following the European laws concerning the treatment of experimental animals. Intraperitoneal pentobarbital (50 mg/kg) was used for anaesthesia; the right femoro-inguinal regions were shaved with hair clippers. Under an operating microscope, we removed 15 right superficial epigastric veins for study, approached through a postero-medial thigh incision (Fig. 1) to minimise the risk of damaging the femoral axis through the classic inguinal incision. By delicate dissection under the operating microscope, at 25 • magnification using two microforceps with blunt tips, the superficial epigastric vein was separated from its
1. Five explanted epigastric veins, isolated over an average length of 9.98 ram, were tied up to avoid shrinkage. These veins were longitudinally cut with microscissors under the operating microscope, blocked with sharp needles on wooden slides to show the inner vascular wall and fixed in a mixture of 0.2% paraformaldehyde and 0.25% glutaraldehyde in 0.1M cacodilate buffer for lh. 2. Five explanted epigastric veins were fixed by immersion in paraformaldehyde and glutaraldehyde soon after being explanted; the vascular lumens of these vessels were washed in phosphate-buffered saline with a Pasteur pipette, and exposed by longitudinal cuts. 151
152
British Journal of Plastic Surgery
3. Five explanted epigastric veins were fixed as for group 2 and transversely cut with a microtome to obtain rings 0.46 mm in average diameter and 0.7 mm long. All the specimens were dehydrated in graded acetone, dried in carbon dioxide and mounted on aluminium stubs with a biadhesive film of graphite paste for scanning electron microscopy (analysis performed by Philips XL30FEG, Eindhoven, The Netherlands).
Results The mean length of the veins was 9.98mm (range: 9.110.5 mm) and the mean diameter of the rings in group 3 was 0.46 mm (range: 0.3-0.6 mm). The five specimens in group 1, investigated at 48 x to 480 x magnification, showed well-preserved endotheliurns, evidence of longitudinal stretching of the vascular wall and an absence of valves (Fig. 2). The five specimens in group 2 showed no longitudinal stretching; each endothelium was covered by a continuous thin layer of coagulated blood without any signs of abrasion on the luminal surface; no valves were detected (Fig. 3). The five specimens in group 3 consisted of transverse sections of veins, lightly collapsed but showing their
Figure ~-Luminal surface of a vein in group 1; the high magnification permits appreciation of the good state of endothelial preservation.
Figure 3 ~ T h e veins from group 2 show a thin layer of coagulated blood covering the endothelium; the continuity of this layer excludes the presence of any valves.
Figure 4~Transverse section through a vein in group 3; the vein wall is slightly collapsed, but the length of the section (0.7 mm) permits examination of the whole intact lumen. complete lumen, segment by segment; no valves were detected (Fig. 4).
Discussion Many haemodynamic flap models have been based on the rat superficial inferior epigastric bundle, including models of arteriovenous fistulae 1 and epigastric free-flap transfer. 2 In 1959 Krogh postulated that valves are the main obstacle to blood flow in experimental arteriovenous fistulae, but did not specifically mention the superficial epigastric vein of the rat. 6 Few studies have attempted to demonstrate the presence of valves inside the veins of rats: some authors have inferred the presence of valves in the lateral thoracic vein of the rat by microangiographic and reverse-flow resistance studies but without morphologically demonstrating the existence of valves. 7 Doubts regarding the presence of venous valves in the lower extremities of rats still persist, s Our morphological study has demonstrated the absence of venous valves in a segment of the superficial epigastric vein of the rat extending from the femoral vein to its bifurcation into medial and lateral branches. Venous valves have a collagen-fibre framework arising from the subendothelial connective-tissue layer. The two valvular faces, axial and parietal, are covered by endothelial tissue in continuity with the walls of the venous lumen. The valvular structure is sufficiently strong to have resisted the different approaches we used to expose the lumen. The veins in group 1 showed a well-preserved internal surface with intact endothelium in each venous segment; any valvular removal would have caused an easily detectable interruption of the endothelial layer. The presence of a continuous thin layer of coagulated blood inside the veins in group 2 is further evidence of the absence of valves. In fact, we can consider this clot as a natural cast of the vein lumen: if a valve had been removed there would have been an interruption of the coagulated layer. To avoid the possibility of removing the valves with the longitudinal incision we prepared transverse sections, 0.7 mm long, of the venous segments in group 3 so that
Do superficial epigastric veins of rats have valves? we could observe their l u m e n s longitudinally within the depth range of the scanning electron m i c r o s c o p e operating at 167 x (Fig. 4). E x a m i n a t i o n o f 15 sections f r o m each venous s e g m e n t did not show any valves. The use o f epigastric veins f r o m a single side in different rats, instead o f both veins f r o m a smaller n u m b e r o f rats, can be justified by the a i m of obtaining results that included as m a n y anatomical variations as possible: anatomic variations are m o r e frequent b e t w e e n different animals than b e t w e e n s y m m e t r i c parts o f the s a m e individual. The absence of venous valves must facilitate circulation in r e v e r s e - f l o w island flap m o d e l s in the rat. E x p e r i m e n t a l h a e m o d y n a m i c results in rat m o d e l s m a y not be directly extrapolated to the h u m a n clinical situation because o f the presence o f valves in m o s t h u m a n veins.
153 4. Petry JJ, Wortham KA. The anatomy of the epigastric flap in the experimental rat. Plast Reconstr Surg 1984; 74:410-13. 5. Chow SP, Chen DZ, Gu YD. The significance of venous drainage in free flap transfer. Plast Reconstr Surg 1993; 91: 713-15. 6. Krogh A. The Anatomy and Physiology of Capillaries. New York, 1959. 7. Mutaf M, Tasaki Y, Fujii T. Expansion of venous flaps: an experimental study in rats. Br J Plast Surg 1998; 51: 393--401. 8. Tanaka Y, Tajima S. The influence of arterial inflow and venous outflow on the survival of reversed-flow island flaps: an experimental study. Plast Reconstr Surg 1997; 99: 2021-9.
The Authors Luigi Valdatta MD, Assistant Professor of Plastic Surgery Alessandro Thione MD, Resident of Plastic Surgery Mara Buoro MD, Resident of Plastic Surgery Angela Faga MD, Associate Professor of Plastic Surgery Plastic Surgery Section Terenzio Congin BD, Assistant Professor of Anatomy Carlo DalrOrbo MD, Professor of Anatomy
References 1. Voukidis T. An axial-pattern flap based on the arterialised venous network: an experimental study in rats. Br J Plast Surg 1982; 35: 524-9. 2. Ruby LK, Greene M, Risitano G, Torrejon R, Belsky MR. Experience with epigastric free flap transfer in the rat: technique and results. Microsurgery 1984; 5: 102-4. 3. Miles DAG, Crosby NL, Clapson JB. The role of the venous system in the abdominal flap of the rat. Plast Reconstr Surg 1997; 99: 2030-3.
Institute of Anatomy University of Insubria, 21100 Varese, Italy. Correspondence to Luigi Valdatta MD, Plastic Surgery Unit, University of Insubria, V. le Borri 57, 21100 Varese, Italy. Paper received 4 January 2000. Accepted 25 September 2000. Published online 5 January 2001.
SURGERY
Do superficial epigastric veins of rats have valves? L. Valdatta, T. Congiu*, A. Thione, M. Buoro, A. Faga and C. Dall'Orbo*
Plastic Surgery Section, and *Institute of Anatomy, University of lnsubria, Varese, Italy SUMMARY. The rat is a good model for experimental studies on the haemodynamic patterns of free-flap transfer but models involving the superficial epigastric vein have not been supported by the demonstration of the anatomical presence or absence of valves inside it. We performed a morphological study to assess whether or not valves are present inside the superficial epigastric vein of the rat. Segments of superficial epigastric veins were explanted from fifteen Sprague-Dawley rats. The lumen was studied with scanning electron microscopy after longitudinal or transverse incision of the venous walls. No valves were detected and we saw no evidence of their accidental removal. As most human veins do have valves, the clinical implications of haemodynamic data on reverse-flow island flaps obtained in rats must be considered very carefully. 9 2001 The British Association of Plastic Surgeons
Keywords: microsurgery, rat, venous valves.
The rat is a useful model for experimental microsurgery; its small size, resistance to infection and limited cost make it very suitable for experimental studies concerning the haemodynamic patterns of free flaps. Haemodynamic models involving the superficial epigastric bundle have been described, 1-3 focusing attention on the vascular anatomy 4 without specifying the presence or absence of valves inside the superficial epigastric vein. The presence of valves could be inferred from the results of experimental studies concerning the perfusion of the arterialised epigastric venous flap in the rat but, as far as we know, no study has clearly demonstrated the anatomic presence of venous valves inside the superficial epigastric venous channel. 5 We performed a morphologic study on the superficial epigastric vein of the rat to investigate the presence or absence of valves. Implications of the data collected for the venous outflow of experimental reverse-flow island flaps in rats are discussed.
I i
SUPERFICIAL EPtr~ TRUNK
Figure 1---The classical inguinal incision (continuous line) and our modifiedapproachthrough a postero-medialthigh incision (dashedline). arterial and nervous counterparts from its origin at the femoral vein to the bifurcation into its medial and lateral branches. The explanted specimens were divided into three groups in the early stages of our investigation:
Materials and methods We performed a study on 15 male Sprague-Dawley rats, weighing 350-400g, caged and fed as usual, following the European laws concerning the treatment of experimental animals. Intraperitoneal pentobarbital (50 mg/kg) was used for anaesthesia; the right femoro-inguinal regions were shaved with hair clippers. Under an operating microscope, we removed 15 right superficial epigastric veins for study, approached through a postero-medial thigh incision (Fig. 1) to minimise the risk of damaging the femoral axis through the classic inguinal incision. By delicate dissection under the operating microscope, at 25 • magnification using two microforceps with blunt tips, the superficial epigastric vein was separated from its
1. Five explanted epigastric veins, isolated over an average length of 9.98 ram, were tied up to avoid shrinkage. These veins were longitudinally cut with microscissors under the operating microscope, blocked with sharp needles on wooden slides to show the inner vascular wall and fixed in a mixture of 0.2% paraformaldehyde and 0.25% glutaraldehyde in 0.1M cacodilate buffer for lh. 2. Five explanted epigastric veins were fixed by immersion in paraformaldehyde and glutaraldehyde soon after being explanted; the vascular lumens of these vessels were washed in phosphate-buffered saline with a Pasteur pipette, and exposed by longitudinal cuts. 151
152
British Journal of Plastic Surgery
3. Five explanted epigastric veins were fixed as for group 2 and transversely cut with a microtome to obtain rings 0.46 mm in average diameter and 0.7 mm long. All the specimens were dehydrated in graded acetone, dried in carbon dioxide and mounted on aluminium stubs with a biadhesive film of graphite paste for scanning electron microscopy (analysis performed by Philips XL30FEG, Eindhoven, The Netherlands).
Results The mean length of the veins was 9.98mm (range: 9.110.5 mm) and the mean diameter of the rings in group 3 was 0.46 mm (range: 0.3-0.6 mm). The five specimens in group 1, investigated at 48 x to 480 x magnification, showed well-preserved endotheliurns, evidence of longitudinal stretching of the vascular wall and an absence of valves (Fig. 2). The five specimens in group 2 showed no longitudinal stretching; each endothelium was covered by a continuous thin layer of coagulated blood without any signs of abrasion on the luminal surface; no valves were detected (Fig. 3). The five specimens in group 3 consisted of transverse sections of veins, lightly collapsed but showing their
Figure ~-Luminal surface of a vein in group 1; the high magnification permits appreciation of the good state of endothelial preservation.
Figure 3 ~ T h e veins from group 2 show a thin layer of coagulated blood covering the endothelium; the continuity of this layer excludes the presence of any valves.
Figure 4~Transverse section through a vein in group 3; the vein wall is slightly collapsed, but the length of the section (0.7 mm) permits examination of the whole intact lumen. complete lumen, segment by segment; no valves were detected (Fig. 4).
Discussion Many haemodynamic flap models have been based on the rat superficial inferior epigastric bundle, including models of arteriovenous fistulae 1 and epigastric free-flap transfer. 2 In 1959 Krogh postulated that valves are the main obstacle to blood flow in experimental arteriovenous fistulae, but did not specifically mention the superficial epigastric vein of the rat. 6 Few studies have attempted to demonstrate the presence of valves inside the veins of rats: some authors have inferred the presence of valves in the lateral thoracic vein of the rat by microangiographic and reverse-flow resistance studies but without morphologically demonstrating the existence of valves. 7 Doubts regarding the presence of venous valves in the lower extremities of rats still persist, s Our morphological study has demonstrated the absence of venous valves in a segment of the superficial epigastric vein of the rat extending from the femoral vein to its bifurcation into medial and lateral branches. Venous valves have a collagen-fibre framework arising from the subendothelial connective-tissue layer. The two valvular faces, axial and parietal, are covered by endothelial tissue in continuity with the walls of the venous lumen. The valvular structure is sufficiently strong to have resisted the different approaches we used to expose the lumen. The veins in group 1 showed a well-preserved internal surface with intact endothelium in each venous segment; any valvular removal would have caused an easily detectable interruption of the endothelial layer. The presence of a continuous thin layer of coagulated blood inside the veins in group 2 is further evidence of the absence of valves. In fact, we can consider this clot as a natural cast of the vein lumen: if a valve had been removed there would have been an interruption of the coagulated layer. To avoid the possibility of removing the valves with the longitudinal incision we prepared transverse sections, 0.7 mm long, of the venous segments in group 3 so that
Do superficial epigastric veins of rats have valves? we could observe their l u m e n s longitudinally within the depth range of the scanning electron m i c r o s c o p e operating at 167 x (Fig. 4). E x a m i n a t i o n o f 15 sections f r o m each venous s e g m e n t did not show any valves. The use o f epigastric veins f r o m a single side in different rats, instead o f both veins f r o m a smaller n u m b e r o f rats, can be justified by the a i m of obtaining results that included as m a n y anatomical variations as possible: anatomic variations are m o r e frequent b e t w e e n different animals than b e t w e e n s y m m e t r i c parts o f the s a m e individual. The absence of venous valves must facilitate circulation in r e v e r s e - f l o w island flap m o d e l s in the rat. E x p e r i m e n t a l h a e m o d y n a m i c results in rat m o d e l s m a y not be directly extrapolated to the h u m a n clinical situation because o f the presence o f valves in m o s t h u m a n veins.
153 4. Petry JJ, Wortham KA. The anatomy of the epigastric flap in the experimental rat. Plast Reconstr Surg 1984; 74:410-13. 5. Chow SP, Chen DZ, Gu YD. The significance of venous drainage in free flap transfer. Plast Reconstr Surg 1993; 91: 713-15. 6. Krogh A. The Anatomy and Physiology of Capillaries. New York, 1959. 7. Mutaf M, Tasaki Y, Fujii T. Expansion of venous flaps: an experimental study in rats. Br J Plast Surg 1998; 51: 393--401. 8. Tanaka Y, Tajima S. The influence of arterial inflow and venous outflow on the survival of reversed-flow island flaps: an experimental study. Plast Reconstr Surg 1997; 99: 2021-9.
The Authors Luigi Valdatta MD, Assistant Professor of Plastic Surgery Alessandro Thione MD, Resident of Plastic Surgery Mara Buoro MD, Resident of Plastic Surgery Angela Faga MD, Associate Professor of Plastic Surgery Plastic Surgery Section Terenzio Congin BD, Assistant Professor of Anatomy Carlo DalrOrbo MD, Professor of Anatomy
References 1. Voukidis T. An axial-pattern flap based on the arterialised venous network: an experimental study in rats. Br J Plast Surg 1982; 35: 524-9. 2. Ruby LK, Greene M, Risitano G, Torrejon R, Belsky MR. Experience with epigastric free flap transfer in the rat: technique and results. Microsurgery 1984; 5: 102-4. 3. Miles DAG, Crosby NL, Clapson JB. The role of the venous system in the abdominal flap of the rat. Plast Reconstr Surg 1997; 99: 2030-3.
Institute of Anatomy University of Insubria, 21100 Varese, Italy. Correspondence to Luigi Valdatta MD, Plastic Surgery Unit, University of Insubria, V. le Borri 57, 21100 Varese, Italy. Paper received 4 January 2000. Accepted 25 September 2000. Published online 5 January 2001.