Development of a psoriatic-like skin inflammation rat model using imiquimod as an inducing agent.

OBJECTIVE: The present investigation was undertaken to develop a psoriatic-like skin inflammation rat model using imiquimod (IMQ) as an inducing agent.
MATERIALS AND METHODS: The hairs of the back dorsal portion of the Wistar rats were removed and 80, 100, and 120 mg of IMQ cream (5% w/w) for 10 consecutive days was applied to different groups of rats. Further, psoriasis area severity index was used for calculating the psoriatic score, which included scoring of erythema, scaling, and thickening. Various biochemical parameters, pro-inflammatory cytokines, vascular endothelial growth factor (VEGF), and histopathological examination were also performed.
RESULTS: The results demonstrated signs of erythema, scaling, and thickening on group applied with 120 mg and 100 mg of IMQ along with ear thickening. Biochemical evaluation revealed a significant increase in the granulation tissue weight followed by significant decrease in the levels of collagen and hexosamine. The antioxidant parameters superoxide dismutase and catalase were found to decline, while nitric oxide and lipid peroxidation were significantly elevated in skin lesions, also supported by increased pro-inflammatory cytokines expression, i.e., interleukin (IL)-1 β, IL-6, IL-17, tumor necrosis factor-α, and VEGF. Histopathological studies revealed a disturbed natural structure along with increased epidermal proliferation, abnormal differentiation with increased number of keratinocytes in the psoriatic skin tissue.
CONCLUSION: From the overall study, we have successfully developed a psoriatic-like skin inflammation rat model for the first time on Wistar strain using IMQ as an inducing agent.

Introduction

Psoriasis is a disorder that occurs due to immune imbalance, which has already been confirmed by a number of researchers since the past two decades. It is considered to be a life-long chronic immune-mediated inflammatory disease, which is identified by patches of thick skin appearing red and is covered with silvery scales that ranges from few patches to almost the entire body surface.[12] It is estimated that more than 7 million Americans and 2%–4% of the population worldwide are affected from psoriasis.[3]

In investigations associated to psoriasis, there are very few available applicable animal models so far. Epidermal hyperproliferation, papillomatosis, altered differentiation, presence of inflammatory cells, and abnormal vascularity are the ideal features of a psoriatic model. Histological characteristics related to psoriasis typically include epidermal hyperplasia along with elongated rete ridges, hypogranulosis, parakeratosis, dermis and epidermis leukocytic infiltration.[4] At present, there are several animal models which were quite similar to psoriasis; however, clinical and pathological resemblance was observed in very few models. Many genetically engineered models which includes transgenic/knockout or induced type model such as xenotransplantation murine psoriasis models have been developed up to date.[5] Xenograft model is one of the psoriatic mouse models, where psoriatic skin (human) is transplanted on immunodeficient mice that closely resemblance of humans psoriasis with respect to its genetic, phenotypic, and immunological characteristics. However, it has several drawbacks such as it is expensive, requires specialized laboratory skills, and time consuming.[6]

Ideal psoriatic animal models must have clinical hallmarks and must represent particular histomorphological patterns of psoriasis, easily reproducible, inexpensive, and ethical.[7] Imiquimod (IMQ), a well-known immune activator, is said to be a ligand for toll-like receptor 7 (TLR7) and TLR8 and has also recently been reported for its psoriasis-like inflammatory effects. The immunemodulatory action of IMQ in inducing psoriasis is observed due to TLR7stimulation on plasmacytoid dendritic cells along with upregulation of Type I interferon pathway. IMQ-mouse model is said to be a suitable experimental model for psoriasis-related studies that presents a typical psoriatic phenotype, which interfere with adenosine receptor signaling pathway and augment inflammation.[8] A study depicted the role of interleukin (IL)-23/IL-17 axis, in IMQ-induced skin inflammation in mice, which was also confirmed through histopathological examination showing similar characteristics to psoriasis. Therefore, IMQ is considered to be very preferable and convenient model to study the mechanistic evaluations related to acute inflammatory response in animal as well as human.[8] In the present study, we have tried to develop a suitable rat model for psoriatic-like skin inflammation using IMQ as an inducing agent, which may prove to be quite important for researchers working in the field of psoriasis.

Materials and Methods

Experimental animals

Healthy rats of Wistar strain (150–200 g) were obtained from the Central Animal House of Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India, having Reg. No.: 92/1999/CPCSEA (dated: April 28, 1999). The animals were maintained and acclimatized for a period of 7 days before experiment by keeping them in standard environmental conditions, namely, 12 h light/dark cycle, ambient temperature (25°C ± 1°C), and relative humidity (45%–55%). The rats were fed with commercially available rat feed and water ad libitum. The protocols involved in the present investigation were approved from Central Animal Ethical Committee of Rashtrasant Tukadoji Maharaj Nagpur University (Letter No.: IAEC/UDPS/2014/38 dated December 22, 2014) and were carried out as per the standard guidelines of National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

Imiquimod-induced psoriatic rat model

Rats of 12–14-week-old were adopted for this study and their hairs were removed by shaving dorsal portion of each rat, while depilatory cream (Veet, Reckitt Benckiser Pvt. Ltd., India) was used to remove the remaining hairs. The rats were divided into four groups (six animals each), where Group I was served as normal control and was applied with vehicle cream (Vaseline). Group II, III, and IV were applied with IMQ 5% w/w cream (Glenmark Pharmaceuticals Pvt. Ltd, Nasik, India) with a daily quantity of 80, 100, and 120 mg, respectively, on the back skin and 10 mg was applied to the right ear for 10 consecutive days.[9]

Scoring severity of skin inflammation and ear thickness

The severity of skin inflammation was measured by calculating the psoriasis area severity index (PASI) where scaling, erythema, and thickening were independently scored from 0 to 4. In this scale, 0 corresponds to none; 1 corresponds to slight; 2 corresponds to moderate; 3 corresponds to marked, and 4 corresponds to very marked.[10] The thickness observed in the right ear of the rats was also measured with the help of a Vernier calliper and was also referred as a measure of skin inflammation.

Biochemical analysis

Rats were euthanized by administering thiopental sodium (65 mg/kg, i.p.) at the end of 10th postoperative day and the psoriatic skin tissues of both dorsal and ear portion were removed and immediately rinsed using tyrode solution. Some portion of the tissues was dried, weighed followed by treatment with 6N HCl on water bath for 24 h to undergo hydrolysis, and the obtained hydrolysate was further subjected to estimations of hexosamine, hydroxyproline, and hyaluronic acid following standard protocol.[111213] Some part of the collected tissue samples was homogenized in phosphate buffer and the supernatant so obtained was used for determination of nitric oxide (NO) adopting the standard procedure given by Green et al.[14] The tissue samples were also subjected to determination of total DNA[15] and protein content.[16] Further, the study also includes antioxidant evaluations, which included lipid peroxidation (LPO), catalase (CAT), and superoxide dismutase (SOD).[17]

Estimation of cytokines and vascular endothelial growth factor

Enzyme-linked immunosorbent assay (ELISA) method was adopted for the estimations of pro-inflammatory cytokines, i.e., IL-1 β, IL-6, IL-17, tumor necrosis factor-α (TNF-α) (Komabiotech, Korea), and vascular endothelial growth factor (VEGF) (RayBiotech) with the help of commercially available ELISA kits on tissue homogenate. The procedure for the following estimation was carried out as per the instructions given by the manufacture.

Histopathological examination

The sample tissues obtained on 10th day of the experiment were immediately fixed in formalin (10%) followed by dehydration in acetone, which were further embedded in paraffin wax and were processed for taking sections using microtome (4 μm thicknesses). The staining of the tissues sections was done using hematoxylin-eosin (1:1) and were subjected to histological examination with the help of a digital Trinocular microscope (Leica DM 2000).

Statistical analysis

All the experimental results are expressed as mean ± standard error of the mean (n = 6), followed by one-way analysis of variance. Newman–Keuls multiple comparisons test was used for assessing the statistical difference between different groups. Graph Pad Prism, version 5 software (San Diego, CA, USA), was adopted for statistical analysis and P < 0.05 was set as significant level.

Results

Structural features of imiquimod -induced skin inflammation

After 6 days of IMQ application, rats from the group applied with 120 mg and 100 mg of IMQ, displayed the signs of erythema, scaling, and thickening, whereas application of 80 mg of IMQ did not show any sign of inflammation [Figures 1 and 2]. The study also involved the measurement of skin inflammation, on right ear as indicated by ear thickness in rats [Figure 3]. A significant increase in the ear thickness was observed on daily application of IMQ to the right ear, which was quite measurable from day 6 onward in the groups treated with 120 and 100 mg of IMQ.

Figure 1

Images of psoriatic skin lesions of dorsal portion on application of imiquimod. (a) Normal control, (b) Imiquimod 80 mg, (c) Imiquimod 100 mg, (d) Imiquimod 120 mg

Figure 2

Effect of imiquimod application on parameters, i.e., erythema, scales, thickness, and cumulative score of psoriatic skin lesions on dorsal portion as indicated by psoriasis area severity index score. Values are mean ± standard error of the mean (n = 6). Where *P < 0.05 versus normal control

Figure 3

Effect of imiquimod application on ear thickness. Values are mean ± standard error of the mean (n = 6). Where *P < 0.05 versus normal control

Biochemical estimations

The results of the biochemical analysis performed in the present study are represented in Table 1. The overall results showed a significant increase in the weight of granulation tissue, while a significant (P < 0.05) decline in total protein and DNA content in granulation tissues of IMQ induced rats was observed, where 120 mg was found to be more prominent as compared to 80 mg, 100 mg, and normal one. The IMQ-induced rats also showed a significant (P < 0.05) decline in the levels of hydroxyproline and hexosamine with a concomitant increase in the levels of hyaluronic acid as compared to normal rats justifying the induction of psoriasis-like inflammation. The results also demonstrated an alteration in the antioxidant status of the IMQ applied rats, where a significant (P < 0.05) increase in the levels of NO and LPO was observed, while a significant (P < 0.05). decrease in the levels of SOD and CAT was noted.

Table 1

Effect on biochemical parameters on application of imiquimod on psoriatic skin lesions of dorsal portion and right ear

	Normal control	PSI induced 80 (mg)	PSI induced 100 (mg)	PSI induced 120 (mg)
Dorsal portion skin lesions
Hexosamine (mg/100 mg of protein)	0.65±0.10	0.47±0.23	0.39±0.17*	0.25±0.11*
Collagen (mg/100 mg of protein)	8.74±0.85	7.56±0.58	2.18±0.20*	2.72±0.25*
Hyaluronic acid (mg/100 mg of protein)	1.69±0.13	1.60±0.27	0.45±0.09*	0.28±0.28*
Total protein (mg/100 mg of tissue)	3.63±0.33	2.12±0.13	1.09±0.18*	0.99±0.16*
Total DNA (mg/100 mg of tissue)	1.49±0.15	1.47±0.33	0.30±0.03*	0.37±0.04*
SOD (U/min/ng of protein)	3.48±0.27	2.85±0.48	0.44±0.15*	0.38±0.13*
Catalase (U/min/ng of protein	3.52±0.24	2.98±0.62	0.66±0.11*	0.46±0.23*
LPO (U/min/ng of protein)	14.73±0.41	17.74±0.52	21.07±0.50*	25.08±0.56*
NO (mole/mg of protein)	0.85±0.09	2.08±0.16*	5.11±0.28*	7.03±0.37*
Right ear skin lesions
Hexosamine (mg/100 mg of protein)	0.43±0.06	0.34±0.11	0.28±0.26*	0.25±0.31*
Collagen (mg/100 mg of protein)	4.99±0.65	3.95±0.24	2.52±0.43*	2.72±0.25*
Hyaluronic acid (mg/100 mg of protein)	0.14±0.03	0.20±0.07	0.38±0.28*	0.55±0.06*
Total protein (mg/100 mg of tissue)	2.26±0.13	1.01±0.09*	1.13±0.08*	0.99±0.16*
Total DNA (mg/100 mg of tissue)	0.47±0.03	0.39±0.02*	0.20±0.03*	0.37±0.04*
SOD (U/min/ng of protein)	2.39±0.06	2.24±0.08	0.29±0.05*	0.19±0.05*
Catalase (U/min/ng of protein	1.62±0.04	1.39±0.12	0.34±0.04*	0.18±0.13*
LPO (U/min/ng of protein)	11.08±0.31	19.17±0.32*	24.00±0.25*	28.28±0.16*
NO (mole/mg of protein)	1.05±0.22	1.91±0.26	3.99±0.18*	6.33±0.57*

*P<0.05 versus normal control. Values are mean±SEM (n=6). SEM=Standard error of mean, NO=Nitric oxide, LPO=Lipid peroxidation, SOD=Superoxide dismutase

Effect on pro-inflammatory cytokines and vascular endothelial growth factor

Figure 4 represents the impact of the IMQ application on pro-inflammatory cytokines and VEGF on skins of the rats. The results depicted a significant increase in the levels of all the tested pro-inflammatory cytokines in the IMQ-induced rats at all concentrations, where significant effect was observed at 100 and 120 mg of IMQ application. Further, the VEGF levels were found to be significantly increased in the IMQ-treated groups at 120 mg.

Figure 4

Effect of imiquimod application on pro-inflammatory cytokines and vascular endothelial growth factor. (a) interleukin-1β, (b) interleukin-6, (c) interleukin-17, (d) tumor necrosis factor-α, and (e) vascular endothelial growth factor. Values are mean ± standard error of the mean (n = 6). Where *P < 0.05 versus normal control

Histopathological studies

Figures 5 and 6 represent the histopathological view of the psoriatic skin lesions (dorsal portion and ear) of the rats administered with IMQ, where epidermal thickening with increased epidermal proliferation and abnormal differentiation were observed along with increased hyperkeratosis with basal cell layer comprising large numbers of keratinocytes.

Figure 5

Histopathological view of psoriatic skin lesions of dorsal portion on imiquimod application. In figure (a) LS of normal control, (b) LS of imiquimod at 80 mg, (c) LS of imiquimod at 100 mg, (d) LS of imiquimod at 120 mg, (e) TS of normal control, (f) TS of imiquimod at 80 mg, (g) TS of imiquimod at 100 mg, and (h) TS of imiquimod at 120 mg

Figure 6

Histopathological view of psoriatic lesions of right ear on imiquimod application. (a) LS of normal control, (b) LS of imiquimod at 80 mg, (c) LS of imiquimod at 100 mg, and (d) LS of imiquimod at 120 mg

Discussion

Psoriasis is characterized mainly by hyperplasia of the epidermis, thickening of the cornified layer, and infiltration of inflammatory cell into dermis/epidermis followed by erythematous plaque formation.[18] Many investigations are underway to develop a suitable and authentic animal model for psoriasis showing pathologically similar characteristics with that of human. IMQ-induced psoriatic model involves a single synthetic innate, which is an Ag receptor ligand[8] and thus, in immunological terms, it may be considered as clean, as it does not require any adjuvants. In the present study, we have selected the Wistar strain of the rats as it is easily available at a very low cost and are also very adaptive in nature as compared to the previously investigated genetically modified[8] mice strains, which are expensive and also requires special attention and care. The present study demonstrated that application of IMQ for 10 successive days of experimental period showed rat's skin closely resembling human psoriasis in terms of skin thickening, scaling epidermal alterations, and erythema. Application of IMQ at 120 and 100 mg showed increased thickening of epidermal layer, erythema, and hyperkeratosis, which reflected in abnormal differentiation of keratinocyte, as a result of increased proliferation and increased expression of cytokine. The study also confirmed ear inflammation, which was evidenced through ear weight and ear thickness on application of IMQ. Further, there was also a significant increase in PASI at 100 and 120 mg of IMQ application; however, at 80 mg of IMQ application; there was no significant increase in PASI.

Collagen is considered to be an important building block for the skin as it is a very important component of connective tissue that maintains the structure and elasticity of skin. The results demonstrated a decrease in the level of collagen (hydroxyproline), whereas an increase in the level of hyaluronic acid was observed on IMQ application. Hyaluronic acid plays a key role in nutrient exchange, water retention, cell mobilization, and cell differentiation.[19] Accumulation of collagen increases the hexosamine levels as hexosamine helps in electrostatic bonding by providing collagen sites.[20] Thus, it may be presumed that application of IMQ to rats resulted in the development of psoriatic-like skin lesions and deformation of normal skin structure, which may be due to the decreased levels of collagen and hexosamine.

Application of IMQ significantly increased the expression of pro-inflammatory cytokines, namely, TNF-α, IL-1 β, IL-6, and IL-17 which co-operatively stimulated the keratinocytes for producing a variety of inflammatory mediators and growth factors that ultimately resulted in fuelling the psoriatic inflammation vicious cycle.[21] An imbalance in antioxidant system is also one of the important characteristic features for pathogenesis of psoriasis.[22] LPO is also said to be one of the major characteristic features in the advanced stage of psoriasis.[23] Therefore, our observation also revealed an increase in the level of LPO followed by decline in the essential antioxidants SOD and CAT, confirming an abnormal antioxidant status during psoriasis. Our observation also showed a significant elevation in the NO level on IMQ application. NO in a certain amount has been reported to participates in normal healing;[24] however, high concentration of NO in relation with elevated expression of inducible NO synthase (iNOS) in psoriasis plaques, and more specifically in keratinocytes,[2526] are mainly concerned with the psoriasis pathogenesis. Therefore, treatment of psoriasis also focuses on reducing the level of NO, which ultimately will reduce the expression of iNOS.

Literature has revealed that overexpression of VEGF results to increased dermis vascularity, which is said to be an important characteristics features of psoriatic plaques.[27] It has been reported that VEGF circulating levels is generally higher in active psoriasis and lowers down during disease remission.[28] Since VEGF plays a significant role in the pathogenesis of the psoriasis, it is considered to be a promising biomarker in understanding the progression of these diseases. The data obtained from the results did showed the increased expression of VEGF on application of IMQ at 100 and 120 mg, which confirmed the pathogenesis of psoriasis. The results from histopathological examination also revealed an increased epidermal layer thickening, hyperkeratosis, and rete-like ridges elongation at 100 and 120 mg of IMQ application. As compared to the previous investigations, the main salient features [Table 2] of our study involve the successful use of a very common strain of rat, which is more cheaper and easier to handle as compared to highly expensive genetically modified mice strain. Further, the availability of large surface area in rats could also prove beneficial in the measurement of severity of inflammation and other analysis. Like previous studies, our study also justified the role of ILs[8] axis in induction of psoriasis.

Table 2

A comparative overview on the previous reports on imiquimod induced mice model with our imiquimod induced rat model

Parameters	Previous reports	Our reports	Key findings
Animal models for imiquimod	Mostly mice have been used	Rats were used	Rats are easy to handle during surgery process
Strain	Genetically modified strains (e.g., BALB/c, C57BL/6)	Wistar rats	Rats strain used here are easily available
Availability and cost	Available at selective animal house and are highly expensive	Easily available at mostly all animal house with very reasonable cost	The common strain of rats can be easily procured at minimum cost from most of the animal house
Dose of imiquimod	60-70 mg of 5% cream	100-120 mg 5% cream	More amount of cream is utilized due to large surface area of skin
Duration of induction	6-8 days	10 days	Takes 2-3 days extra in case of rats for induction
Surface area of psoriatic lesions	Small surface area	Large surface area	Due to large surface area, measurements can be done easily. Further, sufficient amount of tissue is available for various analysis
Histopathological studies and biochemical analysis	Histopathology showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils	Our histopathology also showed increased epidermal proliferation, abnormal differentiation with increased number of keratinocytes	All type of biochemical and histopathological evaluation can be conducted on rat model due to availability of sufficient quantity of tissue because of large surface area
Pathway involved	Observation showed involvement of IL-17 and IL-23 expression	Observation showed elevated expression of IL-1β, IL-6, IL-17, and TNF-α	Most of the studies have confirmed the role of IL-1β, IL-6, IL-17, and IL-23 in pathogenesis of psoriasis

IL=Interleukin, TNF-α=Tumor necrosis factor alpha

Conclusion

Thus, from the overall study, we have successfully developed a psoriatic-like skin inflammation rat model for the first time using Wistar strain. The study also confirmed that IMQ at a dose range from 100 to 120 mg, if applied for 10 successive days could develop psoriasis-like skin inflammation on the Wistar rats. Furthermore, this model could prove very cheaper and suitable for a rapid faster in vivo screening of antipsoriatic drugs under investigation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.