Endogenous TGF-beta 1 and TGF-beta 2 are not essential for epithelialization and neovascularization in the hairless mouse ear wound model.

BACKGROUND AND AIMS: TGF-beta stimulates neovascularization and epithelialization in healing wounds, yet relatively little is known about the mechanisms involved. Using the hairless mouse ear wound model, we studied the effects endogenous TGF-beta 1 and TGF-beta 2 have on epithelialization and neovascularization following the application of neutralizing antibodies. MATERIAL AND METHODS: Forty-three adult male hairless mice had an excisional wound made on the dorsum of each ear. Using vital microscopy, epithelialization and neovascularization were measured every third day until completion. TGF-beta 1 and TGF-beta 2 antibody, control-IgG, or phosphate buffered saline (PBS) were applied to the wounds. RESULTS AND CONCLUSIONS: Excisional wounds treated with anti-TGF-beta 1 and anti-TGF-beta 2, IgGcontrol IgG, and PBS epithelialized in 11.2 +/- 0.5 days (N = 22), 10.9 +/- 0.6 days (N = 17), and 10.6 +/- 0.6 days (N = 15), respectively and neovascularized in 27.9 +/- 0.5 days (N = 17), 27.1 +/- 0.8 days (N = 14), and 26.1 +/- 0.8 days (N = 10), respectively (mean +/- SEM). There were no significant differences in time to epithelialization and neovascularization between the three groups. Furthermore, there were no differences in the average time course of epithelialization and neovascularization between the three groups throughout the healing process. We conclude that endogenous TGF-beta 1 and TGF-beta 2 are not essential for epithelialization and neovascularization in the hairless mouse ear wound model.

Ketanserin accelerates wound epithelialization and neovascularization.

We investigated the acute effects of topical ketanserin, a 5-HT(2) (serotonin) receptor blocker, on wound epithelialization and vascularization with the use of the hairless mouse ear model. Varying concentrations of Ketanserin (0%, 0.2%, 2.0%, 20% weight/volume) were administered to standardized full-thickness skin wounds on the dorsum of the hairless mouse ear immediately after surgery and daily thereafter. With the use of video microscopy and computer-assisted planimetry, vascularization and epithelialization were traced every third day until the wounds were fully healed. Arteriole diameters at selected sites near the skin wound were measured before wound creation and after wounding. It was concluded that topically administered ketanserin significantly accelerates both the vascular (p < 0.001 at 2% and 20% concentrations) and epithelial (p < 0.001 at 20% concentration) rates of wound healing in full-thickness nonpathologic skin wounds. Vasodilation of terminal arterioles was not a major response to Ketanserin. Faster epithelialization was possibly due to direct effect of ketanserin on epithelial cells.

Regional blood flow, plasma volume, and vascular permeability in the spinal cord, the dural sac, and lumbar nerve roots.

OBJECTIVES: This study quantified the regional blood flow (RBF), plasma volume (PV), and the vascular permeability (VP) in the spinal cord, spinal nerve roots, and the dural sac. SUMMARY OF BACKGROUND DATA: Spinal cord hemodynamics have been investigated. However, the full segmental distribution of blood flow to the spinal cord and nerve roots has not been worked out. Vascular permeability of the spinal cord and nerve roots has been investigated but results regarding the spinal nerve roots have not been consistent. METHODS: Eight Labrador dogs were used. Regional blood flow was measured with microspheres. Plasma volume and vascular permeability were assessed by the distribution spaces of radioactively labeled plasma proteins. RESULTS: Regional blood flow was highest in the cervical and lumbar cord. Average (SEM) RBF values of the cord and spinal nerve roots were 10.4 +/- 1.0 and 4.4 +/- 0.5 mL/min 100 g, respectively. Plasma volume of the spinal cord was 0.85 +/- 0.06 mL/100 g and 1.29 +/- 0.17 mL/100 g in the spinal nerve roots. Vessels in the spinal nerve roots had significantly greater permeability to albumin than those of the spinal cord (P < 0.001). CONCLUSIONS: The quantitative flow measurements showed the existence of three main anatomic territories in the cord. The vascular permeability of vessel in the spinal nerve roots appears greater than that of the spinal cord, indicating that spinal nerve roots are located outside the blood brain barrier. Diffusion might be one of the nutritional pathways to spinal nerve roots.

Biofeedback treatment of children and adults with idiopathic detrusor instability.

In adults and children suffering from urge, urge incontinence, frequent voidings, and nocturia, urodynamic examination often confirms the diagnosis of detrusor instability. We assessed the outcome of biofeedback therapy in 15 children aged 6-12 years and 7 adults aged 20-52 years, all with cystometrically proven detrusor instability. The detrusor pressure was visually conveyed to the patient during repeated bladder fillings. The patient was instructed to interrupt detrusor pressure increments by tensing the pelvic floor musculature. None of the children were found completely cured; however, 9 showed a marked decrease in either the number of the extent of symptoms. Two children showed moderate improvement, while 4 remained unaffected by the treatment. One adult was completely cured, 2 improved moderately, and 4 showed no improvement. None of these patients were converted to stable cystometry. Upon termination of the BF treatment a follow-up study was conducted for patients exhibiting pronounced or moderate improvement. The beneficial effect was maintained except in one case.

Comparison of the effects of commonly used wound agents on epithelialization and neovascularization.

BACKGROUND: The primary effect sought with most topical wound therapy is antimicrobial. Topical wound agents are thought to promote normal healing by protecting the wound from infection. In this study, we examined the effect of six commonly used topical wound agents (bacitracin, sodium hypochlorite, silver nitrate, silver sulfadiazine, mafenide acetate, and povidone-iodine) on epithelialization and neovascularization in noninfected wounds. For this study, a new wound model was used in which direct visualization and quantification of wound epithelialization and neovascularization were carried out throughout the entire healing process. STUDY DESIGN: We measured the effect which 500 U per g of bacitracin, 0.25 percent of sodium hypochlorite, 0.5 percent silver nitrate, 1 percent silver sulfadiazine, 8.5 percent mafenide acetate, and 10 percent povodione-iodine had on the rate of wound epithelialization and neovascularization. The agents were applied topically to 99 circular full-thickness wounds (2.25 mm diameter, 0.125 mm depth) created on the dorsum of male hairless mouse ears. This model enabled us to visualize and measure directly wound epithelialization and neovascularization repeatedly throughout healing, using intravital video microscopy and computerized digitized planimetry. RESULTS: Control wounds and wounds treated with silver sulfadiazine (n = 18) and mafenide acetate (n = 14) epithelialized in 7.2 +/- 0.7, 7.1 +/- 0.3, and 7.3 +/- 0.3 days, respectively. This was significantly (p < 0.01) faster than the wounds treated with povidone-iodine (n = 10), sodium hypochlorite, (n = 8), and bacitracin (n = 13). Wounds treated with povidone-iodine epithelialized the slowest (11.8 +/- 0.55 days). Wound neovascularization was completed most rapidly in the groups treated with povidone-iodine and silver sulfadiazine (15.0 +/- 0.4 and 15.3 +/- 0.7 days, respectively). This was significantly (p < 0.05) faster than wounds treated with silver nitrate (n = 15), which neovascularized in 18.4 +/- 0.56 days. One-half of the wounds treated with sodium hypochlorite (eight of 16) did not epithelialize or neovascularize. CONCLUSIONS: The various antimicrobial agents studied in our in vivo model affect wound epithelialization and neovascularization differently. These effects on these two very important aspects of healing should be taken into consideration when indicating a specific agent for treatment of different types of wounds.

The hairless mouse ear: an in vivo model for studying wound neovascularization.

Microvascular ingrowth into damaged tissue is an essential component of the normal healing process. In fact, wound therapy is often aimed at promoting neovascularization. However, little is known about the mechanisms that regulate microvascular ingrowth into a healing wound. This limited knowledge is largely due to the lack of adequate models in which microvascular ingrowth can be quantitatively analyzed throughout the healing process. To address this deficiency, we developed a model in which a wound was created on the ear of the hairless mouse-a well established model for directly viewing and measuring skin microcirculation. While the animals were under ketamine and xylazine anesthesia, 2.25 mm diameter full-thickness wounds were created on the dorsum of hairless mouse ears down to but not including the cartilage (0.125 mm depth). With the use of video microscopy and computer-assisted digitized planimetry, the precise epithelial and neovascular wound edge was viewed and measured regularly throughout healing. Therefore, this model can provide objective data on wound epithelialization and neovascularization throughout healing. This model was used to examine the effect of topical wound agents on epithelialization and neovascularization. Differential effects by these anti-microbial agents on these two processes were observed, which suggests clinical implications for their use.

Direct visualization and measurements of wound neovascularization: application in microsurgery research.

Neovascularization or angiogenesis is an essential yet poorly understood component of the healing process. In wound healing research, there is a lack of models enabling quantitative and continuous measurements of wound neovascularization. The hairless mouse ear wound model permits quantitative measurements of wound epithelialization and neovascularization continuously throughout the healing process. On the ears of male homozygous (hr/hr) hairless mice, standardized circular full thickness dermal wounds are produced; then, using vital microscopy, these two processes are directly viewed and measured at day 0 and every third day thereafter until these are complete. This model system and its application to clinically relevant situations are reviewed.

Urodynamic biofeedback training for children with bladder-sphincter dyscoordination during voiding.

Neurologically normal children with recurrent urinary tract infections (UTIs), night- and daytime wetting, and urge and painful voiding may have staccato voiding due to pelvic floor contractions. The immediate effect of non-invasive urodynamic biofeedback (BF) therapy was assessed using a historical follow-up study in 31 children aged 5-15 years suffering from urodynamically proven overactive urethra during voiding. A long-term follow-up study was performed to investigate whether improvement was maintained. Twenty-four children (77.5%) benefited from the treatment. Of these 16 (51.5%) were cured, while 8 (26%) had a pronounced reduction in their symptoms. Although the flow was normalized in 17 (55%) and nearly normalized in 7 (22.5%), there was no significant correlation between subjective and objective criteria of improvement. Similarly, no relationship was found between the initial urodynamic characteristics and the treatment outcome. During a mean follow-up time of 4 years (range: 1-7.5 years) two of the initially cured patients relapsed. They were recurred with a refresher course. Three had had a single or a few episodes of cystitis in the course of several years. Of the patients with pronounced reduction in their symptoms, three relapsed. A refresher course was attempted in two patients; one was successful. It can thus be concluded that BF is an effective way of treating this disturbance and the beneficial effect is to a wide degree maintained.

[99mTc]diphosphonate uptake and hemodynamics in experimental arthritis: effect of naproxen in the canine carrageenan injection model.

The impact of naproxen treatment on juxta-articular hemodynamics and bone metabolism in experimental juvenile arthritis was studied in the articular carrageenan injection model. Unilateral gonarthritis was induced for 12 weeks in eight dogs receiving naproxen (dosage, 2 mg/kg) and eight controls. Regional blood flow was assessed by the microsphere method, plasma volume by the distribution space of [125I]fibrinogen, and bone metabolism by the 2-h uptake of [99mTc]diphosphonate ([99mTc]DPD). Synovial effusion was less prominent with naproxen treatment as judged by joint fluid volume and pressure. Naproxen reduced the arthritic capsular hyperemia, almost normalized a severe blood flow increase in patella and both juxta-articular epiphyses, ameliorated an expansion of plasma volume in the patella and the distal femoral epiphysis, and normalized an increased [99mTc]DPD uptake in subchondral femoral bone and the tibial cortex. Significantly increased arteriovenous shunting in the arthritic extremity was unaffected by naproxen. The study suggests that long-term cyclooxygenase inhibition offers protection against hemodynamic and metabolic changes in juxta-articular bone secondary to synovial inflammation.

The microcirculation of myocutaneous island flaps in pigs studied with radioactive blood volume tracers and microspheres of different sizes.

In order to further improve the understanding of hemodynamic changes in the immediate postoperative phase after elevation of myocutaneous flaps, regional blood flow and arteriovenous (A-V) shunting were measured in rectus abdominis island flaps in 8 pigs. Radioactive microspheres of two sizes (15 and 50 micron) were used. Approximately half (53.4 +/- 6 percent) of the 15-micron microspheres and one-fourth (24.1 +/- 6 percent) of the 50-micron microspheres entering the flap appeared in the venous outflow. Compared with the control area, A-V shunting was significantly increased in muscle and substantially more pronounced in skin. Nutritional blood flow, total blood flow, and vascular volume were increased in muscle and unchanged in skin and subcutis. The lowest tissue hematocrit of 7 +/- 1 percent was found in skin as compared with a central hematocrit of 35 +/- 2 percent. Tissue hematocrit in flap muscle was decreased to 17 +/- 2 percent when compared with control muscle (22 +/- 3 percent), and the mean transit time for blood was correspondingly decreased. Thus vasodilation provided increased perfusion through muscular capillaries and through A-V shunts. Shunting of 15-micron microspheres appeared to take place not only in skin, but also in subcutis and muscle, which challenges the widespread belief that A-V shunting does not occur in muscle.

Distribution of blood flow in normal and arthritic joints. Role of arteriovenous shunting studied in growing dogs.

Juvenile dog knee with chronic carrageenin-induced arthritis was studied under general anesthesia with 15-microns and 50-microns-sized microspheres (MS) to compare the distribution of absolute and weight-standardized blood flow in normal and arthritic limbs and to localize possible sites of arteriovenous (AV) shunting. Arthritic joints had severe synovial and capsular hyperemia. Absolute as well as standardized blood flow was increased in juxta-articular epiphyses and patella. Shafts were atrophic and had decreased absolute flow but normal standardized flow. However, redistribution of blood flow occurred among regions within the shafts, e.g., in metaphyses away from growth plates. The mean nonentrapment of 15-microns MS was 13.8% in arthritic limbs and 4.2% in control limbs. The uptake of 50-microns MS was lower than that of 15-microns MS in all bony flow compartments due to differences in their rheologic behavior in larger arteries. The relative distribution of 50-microns MS and 15-microns MS varied considerably among regions within bone. Arthritis caused a net shift in the uptake of 50-microns MS relative to that of 15-microns MS from central to subchondral epiphyseal bone, evidencing precapillary vasodilation, but the relationship was strictly unchanged when bones were examined in toto. This result militates against the hypothesis of AV shunting in arthritic bone.

Fuel metabolism in a pig myocutaneous island flap model.

Unilateral denervated myocutaneous island flaps based on the superior epigastric vessels were raised in 24 pigs and the metabolic changes during the first 6 postoperative hours were monitored. Secondary to flap elevation, decreased arteriovenous (A-V) differences in oxygen, glucose, and alanine levels were observed, indicating the opening of A-V shunts and increased arterialization of the venous blood. Venous outflow increased during the first 3 hours, but the A-V differences in all metabolites were constant over the entire 6-hour observation period. Exchange of intermediary metabolites therefore increased within the first 3 hours, after which a steady state was established. The main flap fuels seemed to be fatty acids, muscle proteins, and glycogen, whereas blood-borne carbohydrates and ketone bodies played only a minor role as energy sources. Anaerobic metabolism was increased secondary to flap elevation from 2 to 6 percent as compared with preelevation values. No changes were found in concentrations of plasma catecholamines, which were constantly high. An average weight gain of 3 to 4 percent per hour was equally distributed to skin, subcutis, panniculus carnosus, and muscle. Thus the flap seemed to adapt to the new perfusion pattern within a few hours by a slightly increased anaerobic metabolism, but still with an oxidative metabolism of more than 90 percent.

Arteriovenous shunting is not associated with venous congestion in bone. Knee tamponade studied with 15-microns and 50-microns microspheres in immature dogs.

Blood flow in the hind-limb bones of 8 immature labrador dogs with unilateral knee joint tamponade at 75 percent of the mean arterial pressure was measured with 15-microns and 50-microns microspheres to determine whether or not arteriovenous shunting occurs in bone with venous congestion caused by increased outflow resistance. The intraosseous pressure was 43 percent of the mean arterial pressure in the experimental distal femoral epiphysis versus 14 percent in the control knee (P less than 0.001). No pressure changes were found in the distal femoral metaphysis. Regional blood flow with 15-microns microspheres decreased centrally in the distal femoral epiphysis and increased centrally in the proximal tibial epiphysis. Metaphyseal blood flow was largely unchanged. A net shift in the preferred embolization site of 50-microns microspheres relative to that of 15-microns microspheres from central to peripheral regions occurred within both juxtaarticular epiphyses, indicating arteriolar vasodilation, but the relation between the uptake of the two microsphere sizes was unchanged when the epiphyses and other bony flow compartments were viewed in toto. The result speaks against the hypothesis of arteriovenous shunting in intraosseous hypertension.

Tissue oxygen tension in myocutaneous flaps. Correlation with blood flow and blood gases.

The relationship between tissue oxygen tension (PO2) and blood flow, blood gas tensions, oxygen delivery, and oxygen consumption was investigated in myocutaneous island flaps in 24 pigs. The tissue PO2 was measured by the silicone rubber tonometer technique. Capillary blood flow was increased during the first 3-4 hours after implantation of tonometers and they must, therefore, be implanted 3-4 hours before use. After raising the flap, mean (SEM) total flap blood flow increased from 11.2 (0.8) ml/min to 16.0 (1.3) ml/min within the first three hours. Mean (SEM) subcutaneous tissue PO2 decreased from 9.5 (0.6) kPa to 5.2 (0.7) kPa over six hours after the procedure, whereas muscle PO2, blood oxygen tension, and arteriovenous oxygen difference were unchanged. Hence, both the supply to, and use of oxygen in, the flap increased. Muscular and subcutaneous oxygen tensions were about 3 and 1 kPa, respectively, lower than venous oxygen tension. Tissue PO2 did not correlate with blood flow, blood oxygen tensions, arteriovenous differences in oxygen tension, or with oxygen supply in a cross section of animals. Tissue PO2 seems to reflect accurately the balance between oxygen supply and consumption, which might be the most useful indicator for continuous flap monitoring.

Arteriovenous shunting and regional blood flow in myocutaneous island flaps: an experimental study in pigs.

In eight pigs, total blood flow, regional capillary blood flow distribution, and arteriovenous (AV) shunting were studied during the first 4 postoperative hours after elevation of a myocutaneous rectus abdominis island flap. Capillary blood flow and AV shunting were measured using radioactive microspheres before flap creation and 1 and 4 hours after surgery. Total blood flow, measured continuously as venous outflow, increased in the first postoperative hour (p less than 0.05). Elevation of the flap caused a slight decrease in skin capillary blood flow (p less than 0.05), whereas muscular capillary blood flow increased (p less than 0.01). AV shunting accounted for 50 percent of the total flap blood flow, whereas it was negligible in the abdominal wall prior to flap elevation. Thus stalk blood flow, skin appearance, and skin temperature may be poor indicators of nutritional capillary perfusion. However, the clinical and nutritional consequences of these findings remain to be established.

Technetium 99m diphosphonate uptake and intraosseous hemodynamics during venous congestion in bone. Knee joint tamponade studied in immature dogs.

Venous congestion in bone is a common early feature of inflammatory and degenerative joint diseases. An experimental study was performed of the relationship between the scintigraphic appearance of joints and the intraosseous hemodynamics during venous congestion caused by obstruction of the osseous venous drainage by increased intra-articular pressure. Intra-articular pressure was raised to 75% of mean arterial pressure in one knee each of 8 immature dogs. This caused elevated intraosseous pressure in the distal femoral epiphysis but not in the metaphysis. The elevated intraosseous pressure in the affected femoral epiphysis was associated with decreased technetium 99m diphosphonate uptake and blood flow, unaltered vascular volume and tissue hematocrit, and prolonged transit time of blood in the central cancellous bone. The decreased radionuclide uptake during intraosseous venous congestion thus appeared to reflect a decreased blood supply. However, by multiple regression analysis, the diphosphonate uptake in bone generally varied positively with blood flow and plasma volume and inversely with red cell volume in a nonlinear, multifactorial relationship.

Continuous direct tissue oxygen tension measurement. A new application for an intravascular oxygen sensor.

An oxygen tension measurement equipment, developed for continuous intravascular monitoring was tested as a direct tissue oxygen tension meter. The sterile disposable oxygen probe consists of a Clark type polaro-graphic bipolar electrode placed in a 5 cm long flexible polyethylene catheter (OD 0.55 mm). The probe is easily placed, e.g. in subcutaneous tissue or muscle, with the aid of an introduction needle. In vitro measurements as well as subcutaneous and intramuscular readings were performed. The animals were subjected to various experimental conditions such as different inspiratory oxygen fractions, hypovolemia, and circulatory impairment. Results were comparable to those obtained with previously used and well characterized tissue oxygen tension measurement equipment. The clinical applicability of the oxygen sensor for tissue oxygen measurement was assessed in one patient by 40 hours' surveillance in a free myocutaneous flap. The procedure was simple and implied minimal discomfort for the patient.

The influence of tissue tension on the microcirculation in myocutaneous flaps: an experimental study in dogs.

The effect of increased and decreased uniaxial tissue tension on capillary blood flow, plasma volume, edema formation, and vascular permeability in myocutaneous flaps was studied. A total of 64 equally sized arterialized myocutaneous flaps were outlined on the abdomen of 16 dogs. The four areas on each dog were randomized to either untouched control area, reattachment to normal tension, increased (one and one-half times the original length), or decreased (one-half times the original length). Capillary blood flow measured by the radioactive microsphere technique two hours after operation was decreased in the muscular part of flaps reattached at decreased and normal tension (p less than 0.05). The muscular blood flow was unchanged when flaps were reattached at increased tension and significantly higher than in flaps reattached at decreased tension (p less than 0.05). No differences were found in skin or subcutaneous blood flow. There were no differences in plasma volume, vascular permeability, or total water content between flaps attached under different tensions, whereas the plasma volume was increased in muscle in all three flap types (p less than 0.05) presumably secondary to venous stasis. The study implies that the decreased capillary blood flow seen in the muscular component of a myocutaneous flap in the immediate postoperative period may be counteracted by a somewhat increased reattachment tension.

Microvascular hemodynamics in experimental arthritis: disparity between the distribution of microspheres and plasma flow in bone.

The microcirculation in normal and arthritic juxtaarticular bone was studied in 16 young dogs with carragheenan-induced arthritis of one knee. The regional blood flow was determined by the tissue uptake of intracardially injected 15-microns 141Ce-labeled microspheres, and the microvascular plasma volume was determined by the distribution space of circulating 125I-fibrinogen. Disparities between the distribution of plasma flow and microspheres, introduced by plasma skimming or nonentrapment of spheres in the intraosseous circulation, were estimated by 59Fe-transferrin, a third intravascular tracer, injected as a bolus intracardially and trapped peripherally after 15 sec by prompt circulatory arrest. The tissue uptake of the plasma flow tracer was compared to that of microspheres by the ratio between observed and expected activity of 59Fe-transferrin, the expected activity being calculated from the microsphere distribution. The transferrin and microsphere uptake agreed well in patella, marginal epiphyseal bone, and cortical bone, whereas observed activity of transferrin was twice the expected in central epiphyseal bone, three times higher in marrow, and up to eightfold higher in metaphyses adjacent to growth plates. This discrepancy was significantly greater in arthritic bone when the metaphyses were examined in toto. The microsphere method thus appears to underestimate blood flow to cancellous bone and marrow due to uneven distribution of plasma and formed elements from profound plasma skimming and perhaps also by AV shunting.