Objective: To study the effects and safety of Spongilla Spicules (SS) on rat skin.(The present study was approved by the ethic committee for animal study, Faculty of Medicine, Hunan Normal University.)
Material and Method: An experimental study was conducted on 3 groups of Wistar-Furth adult rats. The first group was applied with SS; the second group with SS in 0.9% normal saline (NSS) and the control group with NSS. These agents were applied on and wiped off the rat skin weekly for four weeks, then the skin biopsies were done. The number of SS spicule and the depth of spicule penetration were examined by scanning electron microscope and by polarized light microscope respectively. Skin histopathology was determined by hematoxylin-eosin staining. The gross skin changes were observed.
Results: Under electron microscopic examination, SS was demonstrated as spicule which was sharp-edged, rod-shaped and smooth surface with approximate 150-300 microns in length and 10-20 microns in diameter. Spicule retention was found in the rat skin lasted until day 3 but was undetectable on day 7. The spicules could be detected deep into stratum basalis. Comparing among three groups, the thickness of epidermis in the second group was decreased with statistically significant difference (p = 0.044) by the end of week 7. The dermal thickness of all groups was increased by age. No any gross skin alteration of all groups was observed.
Conclusion: The authors hypothesized that the spicule causes puncture that enhances penetration into the skin. This solution was safe in the short term usage. However, the long term safety regarding granulomatous formation is still questionable.
Keywords: Spongilla Spicule, Acne vulgaris, Rat skin
Acne vulgaris is a common skin disease of pilosebaceous unit. The etiology is multifactorrial(1). The pathogeneses are follicular epithelial hyper- proliferation, sebaceous gland hyperfunction, inflammation and Propionibacterium acne(1-3).
Antibiotics and retinoids are the standard treatments of acne. Topical therapy alone (benzoyl peroxide, clindamycin, erythromycin, retinoic acid) or in combination with oral therapy (antibiotics, retinoic acid) is recommended according to the severity of acne. Currently, new modalities of acne treatment have been developed including light therapies which are blue light, intense pulsed light and lasers(4,5).
Spongilla Spicules is a freshwater sponge in the phylum of Pohfera where its habitations are in
An experimental study was conducted on 32 Wistar-Furth adult rats from the National Animal Model Center, Hunan Normal University. The age was 8 weeks and the mean starting weight was 258 grams. They were raised in the same condition and categorized into 3 groups. The first group would be applied with SS extract in solution, the second group with SS in 0.9% normal saline solution (NSS) and the control group with 0.9% NSS. The number of rats used in the present study was 15 in first group, 15 in second group and 3 in control groupwith 0.9% NSS. The number of rats used in the present study was 15 in first group, 15 in second group and 3 in control group.
Anesthetic procedures were done by putting the rats into a jar filled with ether. Once the rats were fully unconscious, they would be shaved on week 0 at their backs creating a 10 cm2 area. After that, the prepared solutions were thoroughly applied by massaging in circular motion for 10 minutes and leaved on for another 20 minutes before wiping off with 0.9% NSS. Then one cm2 of rat’s skin biopsy was performed for scanning electron microscopic examination and histological examination with hematoxylin-eosin (H & E) staining and polarized light microscope. The solution application was done every week for 4 weeks and biopsies were performed on day 1, 2, 3, 7, 14, 21, 28, 35 and 49 after solution application and wiping off to evaluate spicule retention and skin.
The amount of spicule retention was studied with scanning electron microscope by counting the numbers of spicule per 1 cm2. Levels of spicule penetration into the skin was determined by using polarized light microscope. The skin histopathology was examined by H & E staining and the thickness of epidermis and dermis was measured. All of the histo- pathological examinations were interpreted by the author number 2 and confirmed by the author number 1 and 3. The gross skin alterations such as erythema, edema and infection were also subjectively observed on day 1, 2, 3, 7, 14, 21, 28, 35 and 49.
The weights, epidermal and dermal thick- nesses of the rat skin were compared between groups by using unpaired t-test with statistically significant difference when p-value was less than 0.05. The gross skin reaction was reported with descriptive method.
From histological examination of the rat skin with H & E staining, the authors found that the stratum corneum of rat skin applied with SS showed more exfoliation than applied with normal saline solution. By using polarized light microscope, the background could be seen in purplish-red and the tip of SS spicule could be seen piercing into stratum basalis (Fig. 2), no spicule was detected in the dermis.
Regarding epidermal thickness, both the experimental and control groups, showed no statistically significant difference from week 0-5. However, by the end of week 7, the epidermal thickness of the SS group was less than those of the SS group and the control group with statisticallysignificant difference (p = 0.044) (Table 2). As the authors observed that rat dermal thickness of both experimental groups showed more significant difference (p = 0.047) than those of the control group on week 7 (Table 3), the authors then statistically evaluated the correlation between rat weight and the dermal thickness in every group. It was found that there was a statistical correlation relevant with R2 = 0.6768 which can be concluded that the increasing in dermal thickness was a result of weight gaining of the rats as they grew up during the experimental time, not a result of the treatment.
Concerning the scanning electron microscope and polarized light microscopic studies on spicule retention, the authors found that the number of spicule were decreased everyday on day 1, 2 and 3. The first group contained less spicule than the second group. Moreover, there was no retention of spicules in both groups on day 7, the number of spicule also decreased each week after reapplying the agent on day 14 and 21. There was no retention of the spicule on day 35 and 49. With gross observation, no skin inflammation (e.g. esiculation, erythema and edema) and infection were detected. The histological examination showed that there was no inflammatory reaction (e.g. spongiotic dermatitis, granulomatous reaction).
The present study of Spongilla Spicules solution on rat skin demonstrated the desquamation of stratum corneum and thinning of the epidermis. Moreover, the increase in dermal thickness correlated with the rat weight gain.
Firstly, the authors hypothesized that the mechanism of actions of SS spicule may be either microablative technique(11) or puncture-resembling mesotherapy due to the small and sharp thorn-like nature of the spicule (150-300 micron in length, which was nearly the size of aluminum hydroxide crystals used in microdermabrasion). But the evidence from previous studies on the effect of microdermabrasion on human skin demonstrated that there was the increment in thickness of epidermis and dermis of the treated skin(12,13). Furthermore, various inflammatory markers of dermal collagen remodeling through wound healing process were also detected e.g. transcription factor AP- 1, NF-Kβ, IL-1β and metalloproteinase 1 and 3(14). In present study, the change in the rats’ epidermal and dermal thickness did not support the mechanism by which microdermabrasion acts on the treated skin. Moreover, the rat epidermis in the SS was decreased; the epidermal thickness of the SS was unremarkable and the rats’ dermis of both groups were unchanged. In addition, the authors detected the exfoliation of stratum corneum. Therefore, the effect of spicule may only cause deeper puncture into the skin hence facilitating hydrogen peroxide to be penetrated deeper into the skin which can eventually eradicate Propionibacterium acne. There was some evidences which demonstrated the role of H2O2 stabilized cream for acne treatment(14,15). It has shown to be as effective as benzoyl peroxide in reduction of both inflammatory and non-inflammatory acne lesions in mild to moderate severity.
Regarding the safety aspect of the SS, the spicule retention was detected after application of the agents in the first 3 days, but after one week, no spicule could longer be detected. This might be due to the wipe-off technique of the procedure that diminished the spicule. Moreover, the authors observed that the spicules pierced into stratum basalis but not into the dermis. This may be according to the application pressure of the author on the rat skin. Mild pressure may contribute to the fact that the spicules were only be detected deep into stratum basalis and if more pressure was applied they may be found deeper into the dermis.
No rash and no histopathological change of the rat skin were demonstrated (e.g. spongiotic dermatitis and granuloma formation). However, if there was any retention of the spicule which its main component is silicon dioxide, granulomatous reaction in dermis might occur both acutely and in long term(16,17). As described in the report of Kenmochi A et al, granulomatous reaction was observed as early as 3 days after indwelling catheter containing silica was removed from the patient(16). Furthermore, there was a report of granulomatous reaction from glass particle retention after 10 years of trauma(17). Since the spicule retention and depth of spicule piercing might depend upon the pressure application of the author and despite the fact that the present study demonstrated no retention and skin alteration both clinically and histologically after 7 week follow-up, they were safe to use in clinical practice with mild and gentle application in short term treatment. The major concern is that the granulonatous reaction from silica can occur as lately as 10 years. So the long term safety is guarded.
In conclusion, the authors hypothesized that spicule of Spongilla Spicules showed effects on the rats’ skin by causing poration-facilitating penetration of hydrogen peroxide to effectively treat acne vulgaris. Spongilla Spicules was safe to be used on the rats’ skin in short term. While the long term safety cannot be concluded particularly on granulomatous reaction.
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