David C. Rice

Type I and Type III Collagen Content of Healing Wounds in Fetal and Adult Rats

Abstract Full-thickness, dermal wounds were surgically created on the dorsa of fetal rats on the 17th day of gestation. The granulation tissue which developed after 2 days (19 days of gestation) was harvested from six to nine animals and pooled and the collagen was extracted with 0.5 M acetic acid and acetic acid plus pepsin. The ratio of type III:type I collagen was estimated from densitometer scans of electrophoretically separated α-chains. Full-thickness (to fascia depth) wounds were also produced on the dorsa of adult rats and granulation tissue which had developed for different periods of time up to 30 days was excised. Relative proportions of type III and type I collagen were assessed in normal and granulation tissues taken from the adult rats. Both fetal and adult granulation tissues have elevated type III collagen content but normal fetal tissue has a much higher content of type III than does normal adult tissue.

Evidence for the efficacy of TRAM flap delay in a rat model

The dilemma in selecting a variant of the transverse rectus abdominis musculocutaneous (TRAM) flap for autogenous tissue breast reconstruction is to balance maximal flap perfusion against the sacrifice of abdominal-wall integrity. A surgical delay has been suggested as one very simple fascia-sparing technique that simultaneously augments flap volume, but no randomized clinical studies have been performed to prove whether there is any benefit whatsoever following such a maneuver. Because large groups of nearly identical subjects may be more readily available for comparison in an animal model, the rat TRAM flap provides an inexpensive method to further investigate the delay phenomenon in this setting. Staged procedures were performed in 15 Sprague-Dawley rats initially causing a delay by division of one or both dominant vessels (cranial epigastric) supplying the rectus abdominis muscles. Either 2 or 4 weeks later, TRAM flaps were elevated based only on the subservient (caudal epigastric) pedicle. A control group of 7 rats with immediate formation of a TRAM flap had a mean survival of 46.3 ± 15.37 percent of their original flap surface area. After a 2-week delay, viability was 50.2 ± 17.54 percent for ipsilateral and 39.4 ± 7.57 percent for flaps with prior bilateral dominant pedicle division. Neither was significantly different from control (p = 0.68 and 0.38, respectively). However, the ipsilateral 4-week delayed group had 67.8 ± 8.96 percent flap survival, which represented a significant enhancement when compared with the undelayed control TRAM flaps (p < 0.02). Corresponding lead oxide microangiographs confirmed dilatation of “choke” vessels connecting the epigastric vessels at their watershed within the rectus abdominis muscle, as well as collaterals circumventing the site of surgical interruption to reestablish the influence of the dominant source pedicle. This evidence proves that the delay phenomenon can be invoked to augment survival of the rat TRAM flap. However, the optimal timing and extent of surgical interruption of collaterals necessary to achieve this objective even in the rat appear to be variable. Caution must therefore be appreciated before extrapolating these data to human applications, particularly since the source vessels to the rat TRAM flap are in no way homologues to those of the human.

Analysis of Collagen Content in the Fetal Wound

The absence of apparent scar formation following the creation of surgical wounds in utero appears to be a phenomenon peculiarly privileged as a sequela of fetal wound healing. Little information exists to explain this disparity from our knowledge of adult wound healing. Therefore, following creation of surgical wounds in fetal rats, at different intervals the healing wounds were harvested and analyzed for collagen content and types. The average proportion of type III collagen was elevated in normal (26.5%) as well as wounded fetal skin (33.8%) when compared with normal levels for the adult (15%). The total collagen content was markedly diminished in the fetal wound. Although embryonal collagen synthesis apparently does exist in fetal reparative processes, the relationship to the lack of gross scarring remains undetermined.

Efficacy of venous supercharging of the deep inferior epigastric perforator flap in a rat model.

Background: An insidious risk with the use of muscle perforator flaps is the possibility of venous outflow compromise. Congestion in deep inferior epigastric perforator (DIEP) flaps in particular is not infrequent. On an empiric basis, their salvage has been accomplished by augmenting venous drainage through alternative outflow tracts. The validity of this clinical maneuver can now best be tested in a rat DIEP flap model. Methods: The rat DIEP flap is a modification of the rat ventral abdomen flap. Flap perfusion can be based on a single rectus abdominis musculocutaneous perforator. No muscle is included with the flap. Three groups of five male Sprague-Dawley rats each were used. The conventional DIEP flap group had only a solitary perforator artery and venae comitantes and served as the control. The contralateral superficial inferior epigastric vein was included with the flap in the other two groups as a distinctly separate venous outflow tract. The latter two groups differed in that the perforator venae comitantes were either retained or deleted. Results: Mean flap survival in the control DIEP flap group was 80.8 ± 16.3 percent. Retention of the contralateral superficial inferior epigastric vein resulted in a statistically significant enhancement in flap viability (p < 0.027) whether the perforator venae comitantes were left intact (99.8 ± 0.4 percent) or had been intentionally ablated (99.6 ± 0.5 percent). Conclusions: Venous supercharging of the rat DIEP flap ensures greater flap survival. As a corollary, this supports the efficacy of prior anecdotal experiences in which an alternative venous outflow tract, preferably from the superficial system, had been used to overcome venous congestion. It is advisable for any muscle perforator flap to always try to retain a second outflow source to allow the potential for venous supercharging, if later indicated.

Restoration of the foot using the radial forearm flap

Large foot defects unsuitable for reconstruction by local foot flaps are most expediently salvaged with distant free-tissue transfers. Although muscle flaps are preferred for infected wounds, coverage of the clean or acute foot deformity may be better achieved with the innervated radial forearm fasciocutaneous flap. This almost ideal donor site has been used by us for all traumatic foot defects requiring free flaps during the previous year. Our results document that in the 5 available clinical examples, restoration of normal foot contour, durability during ambulation, and an excellent aesthetic appearance were achieved.

Objective Monitoring for Safe Tissue Expansion

The high incidence of complications during tissue expansion may be related to overexpansion with subsequent tissue necrosis and implant extrusion. Subjective parameters such as pain or capillary fill may be poor guidelines for determining the end point of a given session of expansion. In an attempt to rectify this problem, we have shown in a prospective study of eight consecutive patients that noninvasive objective monitoring utilizing transcutaneous measured oxygen levels, local perfusion, and implant pressures allowed us to quickly and safely expand each patient. Although perhaps cumbersome for routine use, this system proved valuable in complicated situations where subjective parameters could not be observed.

Comparison of a new method for computer analysis with standard techniques for measuring survival rates in the rat transverse rectus abdominis musculocutaneous flap

Many methods have been used to measure experimental flap survival in animal models. These have previously included planimetry, computer programs, and (routinely by the authors) the simple measurement of the weight of a template traced according to the dimensions of a given flap. To test the validity of their method, an alternative technique was developed by the authors’ biostatiticians. Initially, in a study of rat transverse rectus abdominis musculocutaneous (TRAM) flaps, all templates were also scanned electronically to form bitmaps. Using commonly available software programs, each bitmap was colorized corresponding to viable and nonviable areas of the flaps. A summation of individual pixels by color could then be used to calculate the percentage of flap survival. A comparison of both methodologies in 64 rat TRAM flaps found that the survival rate was slightly greater overall when a computer analysis had been performed (3.1 ± 1.7%), but this was not a significant difference (p = 0.479). Thus, the previous standard method was proven reliable, but just as precise calculations are now possible using the convenience of any personal computer.

Cranial Epigastric Perforator Flap: a Rat Model of a True Perforator Flap.

The major advantage of a true perforator flap is the ability to capture the skin portion of what previously was a musculocutaneous flap, while totally excluding the muscle for function preservation. To understand better the physiology and dynamics of this flap subtype, a comparable and reliable animal model is essential. This has now been accomplished in the Sprague-Dawley rat using the same abdominal skin territory of the standard rat transverse rectus abdominis musculocutaneous flap, but differing in that all rectus abdominis fascial perforators are isolated via an intramuscular dissection back to the cranial epigastric artery source vessel. Hence, this has appropriately been termed the cranial epigastric perforator flap. From a series of eight rats to date, consistent survival of this flap was as predicted. The dissection itself can be somewhat tedious, but it became easier with experience, making this an excellent training model for learning proper technique in the elevation of any true perforator flap.

Venous supercharging augments survival of the delayed rat TRAM flap

Adequate delay of a pedicled transverse rectus abdominis musculocutaneous (TRAM) flap might not necessarily require interruption of the venous system. The retained ipsilateral deep vein of the dominant pedicle could then be used as a secondary outflow source for potential salvage of a congested flap. A venous “supercharged” rat TRAM flap model has been designed to evaluate the efficacy of this maneuver. Seventy-two female Sprague-Dawley rats (CD) were equally divided into two major groups, differing only in whether a delay by division of the dominant cranial epigastric artery had first been performed. An inferior-based TRAM flap (nondominant) was raised for each rat, with three subsets of 12 rats in each group, ie, with the cranial epigastric vein subsequently divided (group control), retained (supercharged), or retained but the inferior pedicle divided (venous flap). Both supercharged subsets had significantly augmented flap survival when compared with flaps in their group raised without the cranial epigastric vein, whether a delay maneuver had (96 ± 6% vs 89 ± 7%; P = 0.012) or not (80 ± 8% vs 65 ± 21%; P = 0.034) been performed. Flaps with only a cranial epigastric vein pedicle totally necrosed, implying that the observed enhancement in flap viability was not the result of transformation into a venous flap, but perhaps as a crossover flap where an adjacent venosome was captured. Venous supercharging can be accomplished by inclusion of the ipsilateral dominant deep vein, and should be a consideration in the clinical planning of delay maneuvers and for treatment of the compromised TRAM flap.

Fate of the TRAM flap after abdominoplasty in a rat model.

&NA; During a classical abdominoplasty, all musculocutaneous perforators from the deep inferior epigastric vessels are normally divided. Even if somehow neovascularization could relink the abdominal skin and rectus abdominis muscles, reestablishing these same discrete perforators would be unlikely because of the barrier effect of the abdominal wall fascia. Therefore, a lower transverse rectus abdominis musculocutaneous (TRAM) flap intuitively should not regain sufficient vascularity for viability after a prior abdominoplasty, and a history of the latter should be expected to be a major contraindication for this procedure. Nevertheless, anecdotal observations of successful lower TRAM flaps following abdominoplasty seem to contradict our basic principles, which may need better further elucidation. Consequently, this two‐stage study in Sprague‐Dawley rats was undertaken, initially performing an abdominoplasty in all rats. This was followed 1 or 10 months later by the creation of an unipedicled superiorly based TRAM flap that incorporated virtually all of the abdominal skin. From our identical historical TRAM flap control (n = 5) except without prior abdominoplasty, 72.8 ± 12.83 percent of this area survived. TRAM flaps raised 1 month after the abdominoplasty (n = 6) had 2.2 ± 3.4 percent or essentially no viability. Unexpectedly, the long‐term group (n = 7) demonstrated 13.7 ± 10.0 percent viability, ranging from 0 to 30 percent. Both groups of TRAM flaps after abdominoplasty had a flap survival area significantly less than that of the control by two‐tailed group t test (p < 0.001), and that of the longterm group area was significantly greater than that of the short‐term (p = 0.022). Lead oxide studies 10 months after abdominoplasty revealed no irrefutable evidence of the reestablishment of rectus abdominis perforators to the integument, although obviously some reconnections had formed at the microcirculatory level to partially revascularize some flaps. The range of viability of the long‐term rat TRAM flaps documented that for the majority, surviving surface area was minuscule even following a delay equivalent to a human decade after abdominoplasty (1 rat month ‐ 1.1 human years), yet rarely sufficient revascularization did indeed occur, which could explain the prior unusual clinical successes. However, the basic principle that a TRAM flap raised following a classical abdominoplasty at any time would be a risky maneuver seems to still be a valid concept. (Plast. Reconstr. Surg. 101: 1828, 1998.)