Similarities between porcine and human skin make the pig an ideal model for preclinical studies of cutaneous inflammation and wound healing. Complete Freund’s adjuvant (CFA) has been used to induce inflammation and to study inflammatory pain in several animal models. Here, we evaluated the inflammation caused by CFA injected in different layers of skin and subcutaneous (SC) tissue in a large-animal model. The degree of inflammation was evaluated at early and late time points by visual inspection and histopathologic analysis. In addition, the side effects of CFA injections were evaluated based on clinical findings, behavioral changes, physiologic state, and (histo)pathologic lesions. Pigs were injected with CFA at the back of the neck’s skin at different depths. All animals showed histologic signs of inflammation at the injection site. Animals injected SC did not show any signs of pain or distress (loss of appetite, abnormal behavior) and did not require pain medication. Inflammation was followed by measuring the area of induration beneath the skin. Animals injected into the dermis and/or epidermis demonstrated a severe inflammatory response on the skin surface with massive swelling, redness within 12hrs of CFA injection, and severe skin necrosis within a week, preventing accurate induration measurements. In contrast to animals injected SC, animals receiving intradermal and/or intraepidermal injection of CFA showed signs of distress requiring pain medication.
Inflammatory skin diseases are the most common problem in dermatology, affecting over 35 million Americans, who spend over $2 billion annually to treat their symptoms (
Animal models of tissue injury and inflammation can be subdivided into those that produce inflammation of cutaneous versus subcutaneous (SC) tissues. Complete Freund’s adjuvant (CFA) has been used to induce inflammation and study inflammatory pain in several animal models [
CFA consists of mineral oil, containing a suspension of whole or pulverized heat-killed mycobacteria [
Inflammatory skin diseases are long-term conditions and are likely to need on-going care, often throughout a patient’s life. By understanding the cellular events that are involved in skin inflammation in a preclinical animal model such as the pig, we could potentially develop therapeutic interventions to treat these diseases.
Here we have tested the feasibility of injecting CFA through different routes as a model of skin inflammation in a miniature swine large-animal model. The aim of this study was to evaluate the relation between the depth of injection and the severity of skin inflammation caused by CFA and to correlate this inflammation with the distress caused to the animals. The results of this study demonstrate that it is feasible to use CFA in pigs in a humane manner without causing alteration of the well-being of the animals if the CFA is injected well below the dermal layer and into the SC space.
Seven MGH miniature swine of either sex, weighing between 35 and 50 kg were used for this experiment. The animals were housed under environmentally controlled conditions and were fed pig diet and water ad libitum. All experiments were approved and performed in compliance with the Institutional Animal Care and Use Committee. Animals were housed at the Center for Transplantation Sciences in accordance with the Guide for the Care and Use of Laboratory Animals. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of the Massachusetts General Hospital.
Complete Freund’s adjuvant (1 mg/ml of mycobacterial components) was used. Prior to injection, animals underwent sedation with 10mg/kg of IM ketamine, followed by maintenance on isoflurane. All injections were accomplished on shaved areas. The skin was cleaned with povidone iodine scrub and 70% alcohol. Two routes of injection were tested: intraepidermal/dermal (n=2) and SC (n=5). One of the animals (pig# 3) underwent 8 different injections using varying needle sizes to test different CFA injection depths into the SC space.
Induction of the inflammatory reaction was achieved by the injection of well resuspended Complete Freud Adjuvant (CFA) without prior emulsification in 4 different areas at the nape of the neck on both right and left side. The pattern of injection was as shown in Figure
Following CFA injection, we used the initial body weight as a reference value, to assess the loss or gain of weight over time, which was represented as a percentage of weight loss. In addition, body temperature, appetite, and behavior were parameters used for the clinical assessment in our animals. Pain and distress were assessed when all injection sites were palpated to subjectively determine whether the manipulation of the lesions caused discomfort to the pigs.
All animals underwent skin biopsy prior to and by day 9 after CFA injection to monitor the levels of evolving inflammation. Two (3-6 mm) punch biopsies were taken from skin surrounding the area of CFA injection on the days mentioned above. Samples were stored in formalin, processed, and stained with H&E. Histopathologic characteristics of skin inflammation were blindly assessed by a pathologist. Additionally, before biopsy, pictures were taken, and redness, induration, and temperature were assessed.
None of the CFA injected animals appeared acutely ill during the study. Animals that received SC CFA injections appeared healthy and did not show signs of pain or distress such as loss of appetite. Regardless of injection route, animals in this study gained weight in a time dependent manner with the exception of SC injected pig # 5 which lost only 0.7% of his initial weight (Table
Table describing the characteristics and demographics of the animals and interventions used.
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Pig #1 | 52.4 | 63.4 | 21% | 4mL | Intraepidermal/25g | Fentanyl patch | No |
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Pig #2 | 48.6 | 53 | 9.1% | 4mL | Intradermal/25g | Fentanyl patch | No |
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Pig #3 | 47 | 52.2 | 11.1% | 4mL | SC diff depths/25g and 27g w/wo 7mm block/4mL | None | No |
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Pig #4 | 46.7 | 57.4 | 22.9% | 4mL | Subcutaneous/25g | None | No |
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Pig #5 | 47.3 | 44 | -0.7% | 4mL | Subcutaneous/25g | None | No |
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Pig #6 | 41 | 47 | 14.6% | 4mL | Subcutaneous/25g | None | No |
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Pig #7 | 37 | 45.1 | 21.9% | 4mL | Intramuscular/25g | None | No |
Macroscopically, inflammation as measured by redness and induration was consistently present at the sites of inoculation, but the severity was dependent on the route and depth of injection as measured by visual inspection (Figure
Early reaction of CFA injection after intradermal injection (a) and SC injection (b). Figure depicts both sites of injection at 1 min and 12hrs after CFA injection.
Intradermal
Subcutaneous
Figure
Macroscopic appearance of CFA injection into different cutaneous spaces at day 9 after injection (a) and histologic evaluation of the inflammatory infiltration (b).
Intraepidermal
Intradermal
SC diff depths
Subcutaneous
Intramuscular
Schematic and real picture representation of the injection sites utilized for CFA administration (a). Schematic representation of the skin layers and thickness to depict the different injections depths used in this model (b).
To determine whether the degree of skin inflammation was related to the injection depth, and not individual animals reacting differently to CFA injection, 4 different SC depths were tested in the same animal in duplicate (Figure
As illustrated in Figure
Macroscopic appearance of the skin following CFA injection into 4 different depths and picture of autopsy depicting the depth of the SC tissue in this particular animal (a). Figures (b–e), macroscopic and histologic appearance of the 4 different injection sites performed at different depths, demonstrating the inflammation in the different layers, epidermis, dermis, and SC tissue.
After injection of CFA using the 25g needle, which is 15.8mm long, the CFA caused massive granuloma formation into the SC space of pigs whose skin thickness ranged between 3 and 4 cm (Figure
Macroscopic appearance and histologic analysis of the granulomas formed at autopsy, when CFA was injected SC (a) and (b) after intramuscular injection.
Unlike skin of rodents, dogs, or nonhuman primates, porcine skin is similar to human skin in terms of structure of epidermal rete ridges, hair follicle structure and density, and presence of sweat glands and subcutaneous fat [
While there have been many reports about the use of CFA in different species, most of them are reported on small animals such as rodents and rabbits [
In this study, we demonstrate that the degree of inflammation and pain affecting the well-being of the animal depends more on the depth of injection than the dose and volume of CFA injected. We used weight loss, diminished food consumption, body temperature, pain, and general condition as indicators of distress in our animals. We injected large amounts of CFA distributed into 4 to 8 sites (each size receiving 0.5ml of 1mg/mL killed mycobacteria), which is two to four times the maximum concentration currently recommended to minimize severe adverse inflammatory reactions [
Given that weight gain is considered an important indicator of animal well-being, the consistent increase between 10 and 20% observed in the initial weight of all animals over the 44-day study clearly suggests that well-being was not compromised (Table
Pain is inferred from scratching behaviors, reduced motor activity of the animal, weight loss, vocalization when the affected site is touched, and a reduction in these behaviors after the administration of opioids. Despite the formation of large granulomas in the deep SC tissue in all SC injected animals, no alteration in behavior, weight loss, body temperature, appetite, pain, or distress were observed. However, this was not the case when animals were injected more superficially (epidermis or superficial dermis) where focal necrosis and ulceration of the skin were observed. These animals showed pain and distress, and although no weight loss was observed, they required pain management and wound care of their skin lesions.
CFA is an oil-based component that when injected diffuses into the surrounding tissue causing an inflammatory reaction. Blood vessels in this respective layer aid in the transport of proinflammatory agents to the circulation. However, in the case of epidermis, a lack of blood supply causes the confinement of CFA and the inability to transport proinflammatory agents, thus a more severe necrosis is observed.
Previous reports have assumed that the development of granulomatous lesions following a SC injection of CFA is painful and distressful for the animals, even though there is no clinical evidence to support this theory [
We found that SC thickness could vary significantly among pigs of similar age, likely due to body score. Pig #7 weighed less than the other pigs, which may have contributed to the unusually thin SC layer in this animal and caused the inadvertent intramuscular CFA injection.
Considering the variations in the pig's skin thickness it would be very helpful to avoid delivery of CFA in an undesired location and therefore avoid compromising the welfare of the animal. Using real-time visualization of needle placement for adequate injection with ultrasound [
Complete Freund’s Adjuvant.
The authors declare that all relevant data supporting the findings of this study are available within the article information files.
The authors declare that there are no conflicts of interest.
Nalú Navarro-Alvarez and Christene A. Huang designed the project; Nalú Navarro-Alvarez, Beatriz M. M. Gonçalves, and Alec R. Andrews conducted experiments; Nalú Navarro-Alvarez, Christene A. Huang, Alec R. Andrews, and David H. Sachs contributed to data analysis; Nalú Navarro-Alvarez wrote the manuscript; and all authors approved the final version.
The authors would like to acknowledge support from CO6RR020135-01 for construction of the facility utilized for production and maintenance of miniature swine. This work was partially supported through a research gift from Eliaz Therapeutics, Inc.