|Year : 2012 | Volume
| Issue : 5 | Page : 629-633
An evaluation of the effects of nonselective and cardioselective β-blockers on wound healing in Sprague Dawley rats
Sanket B Raut, Sharmada R Nerlekar, Sudhir Pawar, Amol N Patil
Department of Pharmacology, L.T.M. Medical College and General Hospital, Sion, Mumbai, India
|Date of Submission||18-Aug-2011|
|Date of Decision||31-Mar-2012|
|Date of Acceptance||01-Jul-2012|
|Date of Web Publication||31-Aug-2012|
Sanket B Raut
Department of Pharmacology, L.T.M. Medical College and General Hospital, Sion, Mumbai
Source of Support: None, Conflict of Interest: None
Objectives: To investigate the effect of a nonselective β-blocker (propranolol) and cardioselective β-blocker (metoprolol) on wound healing in rats using incision and excision wound models and to compare the effect of these drugs on wound healing.
Materials and Methods: Propranolol and metoprolol were given orally. Sprague Dawley rats of either sex were used. Incision and excision wound models were used to evaluate the wound-healing activity. Effects of metoprolol and propranolol on tensile strength, period of epithelialization, and hydroxyproline content were observed. Histological analysis was done to see collagen deposition and inflammatory infiltrate.
Statistical Analysis Used: The data was subjected to analysis of variance (ANOVA) followed by Scheffe's test. P < 0.05 was considered to be statistically significant. Statistical analysis was done using SPSS software version 15.0.
Results: Administration of propranolol or metoprolol was shown to decrease tensile strength, delay wound contraction and re-epithelialization, increase inflammatory infiltrate, and reduce collagen density and hydroxyproline levels.
Conclusions: The results suggest that nonselective and cardioselective β-blockers delay wound healing and these effects are mediated by β1-receptors.
Keywords: Excision wound model, incision wound model, metoprolol, propranolol
|How to cite this article:|
Raut SB, Nerlekar SR, Pawar S, Patil AN. An evaluation of the effects of nonselective and cardioselective β-blockers on wound healing in Sprague Dawley rats. Indian J Pharmacol 2012;44:629-33
|How to cite this URL:|
Raut SB, Nerlekar SR, Pawar S, Patil AN. An evaluation of the effects of nonselective and cardioselective β-blockers on wound healing in Sprague Dawley rats. Indian J Pharmacol [serial online] 2012 [cited 2021 Mar 8];44:629-33. Available from: https://www.ijp-online.com/text.asp?2012/44/5/629/100399
| » Introduction|| |
A wound is the disruption of the normal anatomic structure and, thereby, the function of a tissue.  Wound healing is a physiological process which occurs after injury and can be best described as a complex programmed sequence of cellular and molecular processes which include inflammation, cell migration, angiogenesis, provisional matrix synthesis, deposition of collagen, and re-epitheliazation. ,
Nonhealing ulcers due to angiopathy of diabetes mellitus or vascular disorders like atherosclerosis and varicose veins are very common. In fact, a large number of individuals with indolent or chronic ulcers like pressure sores or decubitus ulcers are often encountered in clinical practice, which substantially increase the cost of therapy. They occur in 3-5% of hospitalized patients.  In addition to the emotional cost associated with the presence of a nonhealing ulcer is the escalating financial burden of the care of these wounds to the patient, to their families, and to society.
The skin is equipped with a dense network of sensory and sympathetic nerve branches distributed in all cutaneous layers. Sympathetic postganglionic fibers maintain cutaneous homeostasis by regulating vasomotor functions, pilomotor activities, and glandular secretion.  Recent studies have shown that the sympathetic nervous system plays an important role in wound healing. ,
Initial studies showed that sympathetic denervation accelerates wound contraction and increases myofibroblast density, but impairs re-epithelialization, inflammation, and mast cell accumulation in rat excisional wounds.  Subsequently, it was shown that mice depleted of norepinephrine by chemical axotomy with 6-hydroxydopamine exhibited reduced re-epithelialization and infiltration of neutrophils and macrophages, and increased angiogenesis.  Recent findings suggest that these effects are mediated by blockade of the β-adrenergic receptors, resulting in aberrant wound healing.  β-adrenoceptors are present in numerous cells that participate in wound healing such as fibroblasts, keratinocytes, mast cells, polymorphonuclear (PMN) leukocytes, and endothelial cells.  In epithelial wound healing, the role of β-adrenoceptors is controversial. Some studies have reported that β-receptor antagonists delay corneal wound healing in rabbits and monkeys,  while others have reported that the same antagonists may accelerate wound closure in bovine and rabbit corneas.  Subsequently, it was shown that oral administration of propranolol (β1 and β2 antagonist) delays wound contraction and re-epithelialization, prolongs the inflammatory phase, and delays granulation tissue formation in rat excisional wound healing.  Nevertheless, activation of the human β2 adrenoceptor delays keratinocyte migration and increases fibroblast proliferation in vitro. 
Furthermore, norepinephrine may suppress the phagocytic efficiency of wound macrophages through both α- and β-adrenoceptor-dependent pathways.  However, the role of each β-adrenoceptor subtype in cutaneous wound healing is still not understood. β-blockers are among the first line of drugs in the treatment of hypertension. But currently, cardioselective β-blockers are preferred by most physicians for the treatment of hypertension. Their effect on wound healing has not been studied previously. Hence we planned this preclinical study to analyze the effect of cardioselective β-blockers on wound healing in rats and compare it with nonselective β-blockers as well as placebo. The objective of the study was to evaluate and compare the effect of cardioselective β-blocker (metoprolol) and nonselective β-blocker (propranolol) on wound-healing activity in rats.
| » Materials and Methods|| |
Institutional Animal Ethics Committee (IAEC) permission was obtained before starting the study. The study was conducted strictly in accordance with the protocol.
Sprague Dawley strain rats of either sex weighing 150-250 g were procured from the Haffkine Institute, Parel, Mumbai and were maintained in standard housing conditions. The animals were fed a commercial diet, and water was provided ad libitum during the experiment. The animals were divided into three groups of six animals each. The animals of group I were left untreated and considered as the control, group II served as reference standard and received metoprolol, and the animals in groups III were treated with propranolol.
Propranolol was administered orally at a dose of 50 mg/ kg 8 with normal saline (obtained from Cipla Ltd). Metoprolol was given orally at a dose of 50 mg/kg 13 with normal saline (obtained from Dr. Reddy's Laboratories Ltd). Administration of the drugs was started one day before wounding. To maintain the adrenoceptor blockade, the drugs were administered daily. Drugs were given with normal saline by gavage feeding. The feeding cannula was inserted in the mouth of the rat in such a way that it reached the esophagus to avoid aspiration. During the procedure of gavage feeding, rats were held in a semierect position in one hand with the tail between the fingers.
Incision and excision wound models were used to evaluate the wound-healing activity.
Incision wound model was selected to study wound healing by primary intention. In incision wound model, under light ether anesthesia, the dorsal skin was shaved and approximately 6 cm long paravertebral incisions were made through the full thickness of the skin on either side of the vertebral column of the rat as described by Ehrlich and Hunt et al. The wounds were closed with interrupted sutures 1 cm apart. The animals were divided into three groups of six animals each. The sutures were removed on the seventh postwound day. The skin-breaking strength of the wounds was measured on the 10th day as described in the method of Lee et al. 
Excision wound model  was selected to study wound healing by secondary intention. Excision wounds were made as described by Morton and Malone.  The rats were inflicted with excision wounds under light ether anesthesia. A circular wound of about 500 sq mm was made on the dorsal thoracic region of the rats. The dorsal regions were shaved, and the surgical area was disinfected with 70% alcohol. A round section of full-thickness skin (diameter of approximately 15 mm) was resected with scissors, and hemostasis was obtained by direct pressure using sterile gauze. The animals were divided into three groups of six each. The drug was given orally, once a day, starting from the day of the operation, till complete epithelialization. The parameters studied were wound closure and epithelialization time. The wounds were traced on mm 2 graph paper on days 4, 8, 12, and 14. The percentage of wound closure was calculated. The period of epithelialization was calculated as the number of days required for falling of the dead tissue remnants of the wound without any residual raw wound.
Estimation of Hydroxyproline Content of the Ulcer
Healed scar on the day of epithelialization was excised and used for determination of hydroxyproline content. 
For histological studies, the wound was excised and fixed in neutral buffered 10% formalin for 24 h and dehydrated with a sequence of ethanol-xylene series of solutions. The materials were infiltered and embedded with paraffin (40-60° C). Microtome sections were taken at 10 μ thickness. The sections were processed in alcohol-xylene series and stained with hemotoxylin-eosin stain, and were observed under a microscope.
All data were expressed as means ± SEM. The data were subjected to ANOVA followed by Scheffe's test. p < 0.05 was considered to be statistically significant. Statistical analysis was done using SPSS software version 15.0.
| » Results|| |
Incision wounds are generally used to study the tensile strength of the wound wherein the sutures are removed on the seventh day post wounding and the tensile strength is measured on 10 th postwounding day. The mean strength of the healed wound scar in the control group of animals was 355.83. The mean strength of the healed wound scar in metoprolol and propranolol group of animals were 256.66 and 236.66, respectively. Both metoprolol and propranolol significantly decreased (P<0.001) the breaking strength as compared to the control. Propranolol decreased the wound-breaking strength significantly more as compared to metoprolol but both decreased it significantly more as compared to the control. The rate of decrease was 28 and 34%, respectively for metoprolol and propranolol [Table 1].
|Table 1: Effect of metoprolol and propranolol on breaking strength in incision wound model (one-way ANOVA)|
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In the excision wound model, the parameters studied were period of epithelialization, hydroxyproline content of the scar, and wound contraction. Period of epithelialization was calculated as number of days required for falling of the dead tissue remnants of wound without any residual scar.
On day 14, in the control group, a small portion (less than 5%) of the original wound remained to be covered by the epidermis. Thus, these animals took a period of 15 days for complete epithelialization. On the other hand, metoprolol and propranolol significantly (P<0.001) decreased the epithelialization process, thus lengthening the period by three and five days, respectively. Propranolol delayed the period of epithelialization significantly more as compared to metoprolol [Table 2].
|Table 2: Effect of metoprolol and propranolol on hydroxyproline content in excision wound model (one-way ANOVA)|
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Estimation of Hydroxyproline
Collagen turnover indicates the grade of healing. The hydroxyproline content in the healed scar of the control group was around 3118 mg/100 g of the tissue. The hydroxyproline content in the healed scar of the metoprolol and propranolol group were around 2733 mg/100 g and 2831 mg/100 g of the tissue, respectively. Propranolol decreased the hydroxyproline content more than metoprolol but both metoprolol and propranolol caused a significant (P<0.001) decrease in the hydroxyproline content of the scar tissues as compared to control [Table 2].
Wound contraction generally occurs in large surface wounds. The contraction helps to close the wound by decreasing the gap between its dermal edges and by reducing the wound surface area. Hence, it is an important feature in healing by secondary union. In the control group, the wound contracted to the extent of 24, 58, 87, and 95% by days 4, 8, 12, and 14. Wounds of the animals, which received propranolol showed a significant decrease in wound contraction as compared to metoprolol on days 4, 8, 12, and 14 but animals which received metoprolol showed a significant (P<0.001) decreased rate of contraction as compared to control on days 4, 8, 12, and 14. Propranolol also showed significantly (P<0.001) lesser wound contraction as compared to control on days 4, 8, 12, and 14 [Table 3].
|Table 3: Effect of metoprolol and propranolol on wound contraction in excision wound model (one-way ANOVA)|
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The histological profiles of the granulation tissue of propranolol-treated animals [Figure 1]c showed more macrophages and less collagenation. On the other hand, the features of the sections of control wounds [Figure 1]a were those of advanced healing, as compared to those of propranolol and metoprolol. These sections of granulation tissue of the control animals showed signs of tissue repair with increased collagen formation [Figure 1]a and less inflammation. However, in metoprolol-treated animals, the healing activity was comparatively lesser with moderate collagenation and retention of the inflammatory infiltrate as compared to control [Figure 1]b.
|Figure 1: (a) Histological section of 15-day-old excision wound of the control group of animals showing few a macrophages with signifi cant collagen deposition (H&E stain). (b) Histological section of 15-day-old|
excision wound of metoprolol group of animals showing moderate collagen deposition with macrophages (H&E stain). (c) Histological section of 15-day-old excision wound of propranolol group of animals
showing more aggregation of macrophages with few collagen fi bers (H&E stain)
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| » Discussion|| |
We studied the role of β-adrenoceptor subpopulations with incision and excision wound models in rat cutaneous wound healing. We observed that propranolol (β1 and β2 antagonist) and metoprolol (β1 antagonist) increased the inflammatory infiltrate, but reduced wound contraction, re-epithelialization, and collagen deposition.
The incision wound model was selected to study wound healing by primary intention. This was done by measurement of the tensile strength. Both metoprolol and propranolol significantly decreased the tensile strength as compared to control. These observations may be explained by previous studies where isoproterenol (a nonselective β-adrenoceptor agonist) increased the proliferation of cultured embryonic murine fibroblasts.  Thus, administration of a β-adrenoceptor antagonist decreased the tensile strength probably by decreasing fibroblast concentration.
Recently, it has been shown that epithelial healing of incisional and excisional lesions is impaired in rats submitted to sympathectomy.  In vitro, isoproterenol (a nonselective β-adrenoceptor agonist) reduces the rate of cell proliferation of mouse and human epidermal keratinocytes through the activation of adenyl cyclase, leading to an increase in intracellular cyclic adenosine monophosphate (cAMP), and propranolol treatment increases the rate of cell proliferation in cultured adult murine keratinocytes.  Therefore, the β1 and β2 adrenoceptor blockade by propranolol delays formation of the neoepidermis, possibly via inhibition of adenyl cyclase and a reduction of cAMP production by keratinocytes.
In rodent models, myofibroblast-mediated contraction is the major mechanism of wound contraction.  Myofibroblasts reduce dermal defects and deposit extracellular matrix (ECM). When re-epithelialization is complete, myofibroblasts undergo death by apoptosis.  The administration of propranolol or metoprolol reduces wound contraction after wounding. Because the interaction between myofibroblasts and the surrounding ECM plays an important role in wound contraction, we speculate that β1 and β2 adrenoceptor blockade delays myofibroblast differentiation and collagen fiber deposition, disturbing the myofibroblast-ECM interaction and delaying wound contraction. Similar results were observed in a previous study of propranolol-treated animals in which myofibroblastic differentiation was delayed and collagen deposition reduced, leading to a delay in wound contraction 14 days after wounding. 
In the present study, both propranolol and metoprolol increased inflammatory infiltrate after wounding [Figure 1]b,c. In vitro models have shown that β-adrenoceptor stimulation with adrenaline and isoproterenol inhibits chemotaxis of rabbit PMN, possibly by reducing cAMP levels.  Therefore, β1 and β2 adrenoceptor blockade increased the migration of PMN, possibly via a reduction in cAMP levels, leading to delayed wound closure.
Administration of propranolol and metoprolol reduced the density of collagen fibers [Figure 1]b,c and the levels of hydroxyproline 14 days after wounding. However, sympathetic denervation increased myofibroblast density and induced the formation of a denser and more mature collagen scaffold.  These observations may be explained by previous studies where isoproterenol (a nonselective β-adrenoceptor agonist) and ZD (a β3-adrenoceptor agonist) increased the proliferation of cultured embryonic murine fibroblasts.  The activation of β3 adrenoceptors on these fibroblastic cells could activate phospholipase C (PLC) and protein kinase C (PKC), increasing the cytoplasmatic calcium concentration and inhibiting the activation of adenyl cyclase stimulated by prostaglandin E2 (PGE2), decreasing the cAMP production.  Moreover, it has been described that propranolol has a low antagonist potency against β3 adrenoceptors  and inhibits hydroxyproline accumulation in cultured human skin fibroblasts.  Thus, the β1 and β2 adrenoceptor blockade may increase myofibroblast density through the stimulation of β3 adrenoceptors; however, the β1 and β2 adrenoceptor blockade could reduce contractile force and ECM deposition by myofibroblasts.
In conclusion, the present study has demonstrated that the administration of propranolol or metoprolol delays wound contraction and re-epithelialization, increases inflammatory infiltrate, and reduces collagen density and hydroxyproline levels. Therefore, the blockade of β1- and β2 adrenoceptors is involved in the alterations caused by sympathetic denervation on excisional cutaneous wound healing. As wound healing is delayed in metoprolol-treated animals, although to a lesser extent as compared to the propranolol-treated group, it seems that most of these effects are mediated by β1 receptors. This information should be considered by physicians during the treatment of patients who present with hypertension and concomitant problems in the healing process.
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[Table 1], [Table 2], [Table 3]
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