Normal Rat Skin Research Articles

Synthesis of new heterocyclic derivatives (benzodioxane-2-yl)methanol and their effect on rat intraocular pressure

Objective: Synthesis of new compounds, melatonin isosters, and study of their ability to reduce intraocular pressure.
 Materials and methods: Based on the previously made forecast and on the similarity with the selected compounds, the synthesis of new compounds was carried out – melatonin isosters having a 2,3-dihydro-1,4-benzodioxin cycle connected by a linker of various chemical structure with a nitrogen-containing heterocyclic core. The structure of the synthesized compounds was proved by 1H- and 13C- NMR spectroscopy, purity and individuality – by chromatographic methods. Intraocular pressure in laboratory animals was measured using a contact veterinary tonometer Tonovet (Finland). Cytotoxicity was assessed using MTT test on normal rat skin fibroblasts.
 Results: Derivatives (benzodioxane-2-yl)methanol reduced the level of IOP in varying degrees of severity. The VMA-22-37 compound showed the greatest activity. With topical application of a 0.4% solution, the compound reduced the intraocular pressure of intact rats by 26.6%, while not affecting the control eye. This compound also has low cytotoxicity.
 Conclusion: Derivatives (benzodioxane-2-yl)methanol is promising for the search for compounds capable of reducing increased intraocular pressure.

α-Terpineol loaded, electron beam crosslinked polyvinyl alcohol/tapioca starch hydrogel sheets; fabrication, characterization and evaluation of wound healing potential on a full thickness acid burn wound.

The multifaceted challenges in treating full-thickness acid burn wounds including impaired tissue regeneration, increased risk of infection, and the pursuit of functional and aesthetically pleasing outcomes, highlights the need for innovative therapeutic approaches for their treatment. The exceptional biochemical and mechanical properties of hydrogels, particularly their extracellular matrix-like nature and their potential to incorporate functional ingredients positions them as promising materials for wound dressings, offering a potential solution to the complexities of full-thickness burn wound management. The current study has integrated functional ingredients (starch and α-terpineol), known for their angiogenic, fibroblast-adhesive, and anti-inflammatory properties into an α-terpineol loaded, electron beam crosslinked polyvinyl alcohol/tapioca pearl starch hydrogel. The hydrogel was then explored for its efficacy in treating full-thickness acid burns. The hydrogel sheets, fabricated using a 25 kGy electron beam, were characterized for structural and functional properties. Surface morphology, gel fraction, swelling ratio, moisture retention capacity and thermal stability were also evaluated. PVA/tapioca starch hydrogel demonstrated optimal macroporosity, mechanical strength, thermal stability, water retention, and moisturizing ability, making it ideal for the intended application. In vitro skin compatibility analysis of α-terpineol-loaded hydrogel confirmed its biocompatibility, demonstrating 90% fibroblast viability. In vivo sensitivity testing on normal rat skin showed no inflammatory response. Analysis of the full-thickness rat chemical burn wounds treated with the hydrogels demonstrated that α-terpineol (AT) loaded e-beam crosslinked PVA/tapioca starch hydrogels increased the rate of wound closure, promoted re-epithelialization, facilitated collagen deposition, stimulated angiogenesis, and promoted keratin deposition, ultimately leading to healing of both thick dermal and epidermal tissues, as well as partial restoration of skin appendages over a duration of 30 days as confirmed by the histological and immunohistochemistry staining. Collectively, this study indicates that α-terpineol (AT) loaded e-beam crosslinked PVA/tapioca starch hydrogel holds promise as a cost-effective and efficient wound dressing for expediting full thickness acid burn wound healing, thus expanding the practical applications of the natural polymer based sheet hydrogel dressings.

Phytochemical screening and wound healing effect of Alocasia longiloba Miq. petiole ethanolic extract and its fractions in excision wound rat model

Alocasia longiloba Miq., or ‘keladi candik’ is traditionally used for wound healing in Malaysia. Preliminary studies have been performed to determine the dose range of A. longiloba for potential wound healing. Therefore, this study continues to determinethe phytochemical screening of fractions and their ability in accelerating wound healing. Ethanol crude extract of A. longiloba and its fractions of hexane, chloroform, and ethyl acetate was prepared and used for phytochemical screening and wound healing formulation. Seven groups of Sprague-Dawley rats (n = 3) were experimentally wounded in the dorsal area for 12 days. Group I was left untreated, Group II was dressed in petroleum jelly as the vehicle group, Group III was treated with Solcoseryl jelly 10% as the positive drug, and the rest of the groups were dressed with respective formulations at 6% concentration. The wound area was photographed and measured, and the histological examinations of granulation tissues were examined. The phytochemical screening of chloroform fraction shows an abundance of alkaloids, flavonoids, and tannins, which are considered responsible for wound healing activity. The chloroform fraction proved more effective, showing a significant percentage of wound contraction (p<0.05) compared to the negative and vehicle control groups. Moreover, histological observation of wounds treated with chloroform fraction of A. longiloba showed an organized epithelial layer, fewer blood vessels, and dense collagen fibres comparable with normal rat skin.

Histopathological Evaluation of the Effects of Low-level Laser Therapy on the Healing of Experimental Cryosurgical Wound in Normal Rat Skin

Introduction: This research aimed to study the healing process of cryosurgical wounds after topical application of low-level laser and to evaluate its effects for the prevention of healing complications. Materials and Methods: A total of 50 healthy male Wistar rats with an average weight of 200-250 g were selected and randomly divided into two main groups. In control group (N=25), cryosurgery was done without using of low-level laser. In experimental group (N=25), cryosurgery was done and the low-level laser was used for 10 days post-surgery. Each main group was divided into five subgroups (n=5) according to histopathological evaluation date (3, 7, 14, 21 and 28 days post-surgical) and healing process was evaluated on the wound specimens harvested at above-mentioned days. The full- thickness wounds were harvested in 40 mm in length and 20 mm in width and the specimens were stained using hematoxylin- eosin and Masson’s Trichrome staining. The healing process was evaluated and compared in groups by observation of granulation tissue and collagen fibers amounts, epithelial gap size, the number of inflammatory cells, and the rate of angiogenesis. Data were collected and statistically analyzed and compared between the experimental and control groups on specific days using the Kruskal-Wallis and Dan post hoc statistical tests. Results: The obtained data were compared between the experimental and control groups on different days using the Kruskal-Wallis and Dan post hoc statistical tests. The statistical results revealed significant differences between groups (P<0.05) in all mentioned variables. It revealed that the healing process in the experimental group took place at least one week earlier than the control group. Conclusion: The results of this animal study showed that the low-level laser therapy on an experimental cryosurgical wound not only accelerates the healing process but also can be effective in the prevention of healing complications. The authors suggest further research to extend the results to human applications.

The Evaluations of Menthol and Propylene Glycol on the Transdermal Delivery System of Dual Drug-Loaded Lyotropic Liquid Crystalline Gels.

This study aimed to evaluate the effects of two different structural alcohol permeation enhancers (menthol and propylene glycol) on the internal structure and in vitro properties of the dual drug-loaded lyotropic liquid crystalline (LLC) gels. The LLC gels were prepared and characterized by polarized light microscopy, small-angle X-ray scattering, differential scanning calorimetry, attenuated total reflectance-Fourier transform infrared spectrum, and rheology. Based on the results, the inner structure of the gels was QII mesophase and exhibited a pseudoplastic fluid behavior. The level of internal order in the LLC mesophase would be affected by introduced 2wt% menthol (MEN) and propylene glycol (PG). The in vitro release experiment showed that the release behavior of sinomenine hydrochloride (SH) and cinnamaldehyde (CA) from the LLC system was dominated by Fickian diffusion (n < 0.43). MEN and PG had the opposite effects on the release of hydrophilic SH, while the MEN and PG both increased the release of lipophilic drug CA. Furthermore, in vitro permeation studies indicated that MEN and PG could both improve the skin permeability of SH and CA, and MEN displayed more pronounced enhancement. All the samples showed no skin irritation on the normal rat skin. Collectively, in our research, monoterpenoid MEN exhibited a better penetration-promoting effect than straight-chain fatty alcohol PG on the dual drug-loaded LLC system.

Effect of Hydrogen Sulfide on the Mitogen-Activated Protein Kinase Signaling Pathway in Cultured Skin Macrophages of Burned Rats

BackgroundThis study aims to investigate the effect of hydrogen sulfide on the mitogen-activated protein kinases signaling pathway in in vitro cultured skin macrophages of burned rats. Materials and methodsThirteen healthy Sprague–Dawley rats were divided into five groups: normal control group, burned control group, sodium hydrogen sulfide group, glibenclamide group, and sodium hydrogen sulfide + glibenclamide group. The burned rats were made into a deep II° 5% total body surface area flame burn injury model. The skin basement macrophages were separated from the skin of normal rats and the wound skin of burned rats and cultured. At 1, 6, and 12 h after intervention, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 protein levels were detected by Western blot, and ERK, p38, and JNK messenger RNA (mRNA) levels were detected by reverse transcription polymerase chain reaction. ResultsDifferences in ERK, p38, and JNK mRNA and protein levels between the normal control group and burned control group were statistically significant (P < 0.05). At the same time point, the ERK, p38, and JNK mRNA and protein levels in the NaSH group were different from those in other groups, and the differences were statistically significant (P < 0.05). ConclusionsHydrogen sulfide has a regulatory effect on ERK, JNK, and p38 in the mitogen-activated protein kinases signaling pathway in macrophages of burned rats.

Involvement of RNA helicase p68 in skin wound healing process in rats

PurposeRNA helicase p68 plays an important role in organ development and maturation through tuning cell proliferation. However, the character and role of p68 in the whole wound healing process need more study. MethodsFirst, we characterize expression of p68 in normal rat skin development postnatal. Then, we assayed dynamic change of p68 in rat skin from different stage after injury, and explored the role of p68 in proliferation and migration of three types of wound healing related cells. Resultsp68 was down-regulated during skin developmental and maturation process, up-regulated after wound, peaked on day 14 and then significantly decreased. Wound fluid enhanced wound healing related cell proliferation and up-regulated expression of p68. Conversely, reducing p68 expression by RNA interference resulted in significantly slower proliferation and migration. ConclusionOur results define an important role of RNA helicase p68 in skin wound healing process.

Annexin A2 functions downstream of c‑Jun N‑terminal kinase to promote skin fibroblast cell migration.

Delayed healing of skin wounds is one of the outcomes of diabetes mellitus (DM), a condition that affects a significant number of patients worldwide. However, the underlying mechanisms remain unknown. In order to examine proteome alterations in DM, a rat model of type 1 diabetes was developed using streptozotocin injections. The proteomic responses of normal and DM rat skin were analyzed by two‑dimensional electrophoresis, and differentially expressed proteins were identified using a liquid chromatography/mass spectrometry system. DM induced 36 and repressed 41 differentially expressed proteins, respectively. Altered proteins were involved in a number of biological processes, including RNA and protein metabolism, the tricarboxylic acid cycle, glycolysis, cytoskeleton regulation, hydrogen detoxification and calcium‑mediated signal transduction. In addition, overexpression of annexin A2, one of the signaling proteins altered by DM, accelerated the rate of human skin fibroblast cell migration. Application of SP600125, an inhibitor of a key regulator of cell migration c‑Jun N‑terminal kinase (JNK), inhibited the migration of normal cells. By contrast, SP600125 treatment did not inhibit the migration of annexin A2‑overexpressed cells, indicating that annexin A2 may function downstream of JNK. In conclusion, the results of the present study reveal the potential proteomic responses to DM in skin tissues, and demonstrate a positive functional role of annexin A2 in fibroblast cell migration.

HII mesophase as a drug delivery system for topical application of methyl salicylate

The main objective of this study was to develop reversed hexagonal (HII) mesophase for transdermal delivery of methyl salicylate. The formulation was prepared, characterized and evaluated for its skin penetration in vitro and skin retention in vivo. Preliminary pharmacodynamics and skin irritation were also investigated. The formulation was identified as hexagonal structure. In vitro study exhibited that HII mesophase enhanced the skin permeation by delivering 2.61 times more methyl salicylate than the commercially available cream. Meanwhile, HII mesophase presented higher bioavailability as AUC(0–24) and AUC(0–∞) were 32.894μg·mL−1 and 32.935μg·mL−1 respectively, while the cream were 12.791μg·mL−1 and 12.970μg·mL−1. Preliminary pharmacodynamics studies demonstrated that HII mesophase possessed anti-inflammatory and analgesic effects for inhibiting paw edema, granuloma and pain. MeSa HII mesophase showed no skin irritation on the normal rat skin. Thus, HII mesophase was considered as an effective delivery system for MeSa.

Development of bioresorbable bilayered systems for application as affordable wound dressings

The objective of this work was the preparation and evaluation of a bioresorbable bilayered system for application in the treatment of dermal lesions. The system was based on a polyesterurethane as the external layer and a gelatin membrane as the internal layer. The polyesterurethane was synthesized from poly(ε-caprolactone), polyethylene glycol of 1 or 10 kDa as a hydrophilic component or Pluronic F127 as an amphiphilic component and l-lysine ethyl ester diisocyanate as an urethane precursor. Gelatin membrane was obtained by crosslinking with the naturally occurring crosslinker genipin. Three important points were addressed in this study: the physicochemical characterization of the system, the in vitro behaviour and the in vivo performance on a full-thickness wound defect of rat. The polyesterurethane containing polyethylene glycol of 10 kDa presented the optimum properties for the designed application as to be tested in animal experiments. The in vivo results showed good healing of the lesion with the formation of epidermis similar to normal rat skin. These promising results suggest the potential of this system to be used as an affordable wound dressing in the treatment of different dermal lesions.

Enhancing Protoporphyrin IX induced Photodynamic Therapy with a Topical Iron Chelating Agent in a Normal Skin Model

Protoporphyrin IX (PpIX)-induced photodynamic therapy (PDT) is being utilised within dermatological practice as a topical method of localised ablation of nonmelanoma skin cancer/precancer. Standardised protocols have been implemented to good effect when the disease remains superficial but improvement is required to widen the application of this light activated drug therapy to treat thicker or acrally located conditions. As innate haem biosynthesis is exploited to accumulate the light sensitive PpIX from a topically applied inert prodrug (aminolaevulinic acid; ALA), this pathway can be further manipulated through the concurrent administration of an iron chelating agent to hyper-accumulate PpIX by temporarily reducing its iron dependent conversion to haem. A topical preparation of ALA was applied to normal rat skin with or without the hydroxypyridinone iron chelator, CP94. Image analysis quantification of tissue fluorescence following excision indicated that ALA plus CP94 produced 29.0% more fluorescence than ALA alone (p < 0.09), peaking at 5 hours. Furthermore, fluorescence spectroscopy of frozen skin samples from each treatment group were characteristic of PpIX (maxima 636 +/- 2 nm), indicating that topical CP94 administration elevated PpIX levels without significantly producing any other fluorescent species. When PDT efficacy was considered post irradiation, a substantial three-fold increase in effect was observed 4 days after treatment when the iron chelator CP94 was co-administered topically with the prodrug (p < 0.07). It has therefore been established that the hydroxypyridinone CP94, is topically active within normal rat skin, effectively chelating iron to elevate PpIX accumulation and thus improve PDT efficacy.

Correlation between polarization sensitive optical coherence tomography and second harmonic generation microscopy in skin.

Both polarization sensitive optical coherence tomography (PS-OCT) and second harmonic generation (SHG) microscopy are 3D optical imaging methods providing information related to collagen in the skin. PS-OCT provides birefringence information which is due to the collagen composition of the skin. SHG microscopy visualizes collagen fibers in the skin based on their SHG property. These two modalities have been applied to the same skin pathologies associated with collagen changes, but their relationship has not been examined. In this study, we tried to find the relationship by imaging the same skin samples with both modalities. Various parts of the normal rat skin and burn damaged skin were imaged ex vivo, and their images were analyzed both qualitatively and quantitatively. PS-OCT images were analyzed to obtain tissue birefringence. SHG images were analyzed to obtain collagen orientation indices by applying 2D Fourier transform. The skin samples having higher birefringence values had higher collagen orientation indices, and a linear correlation was found between them. Burn damaged skin showed decreases in both parameters compared to the control skins. This relationship between the bulk and microscopic properties of skin may be useful for further skin studies.

High-glucose inhibits human fibroblast cell migration in wound healing via repression of bFGF-regulating JNK phosphorylation.

One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression.

Topical application of the synthetic triterpenoid RTA 408 activates Nrf2 and induces cytoprotective genes in rat skin

RTA 408 is a member of the synthetic oleanane triterpenoid class of compounds known to potently activate the cytoprotective transcription factor Nrf2. Because skin is constantly exposed to external oxidative stress, such as that from ultraviolet radiation, from chemical exposure, during improper wound healing, and throughout the course of cancer radiation therapy, it may benefit from activation of Nrf2. This study was conducted to evaluate the transdermal penetration properties and Nrf2 activation potential of RTA 408 in normal rat skin. RTA 408 (0.1, 1.0, or 3.0%) was applied topically to the shaved skin of male Sprague-Dawley rats twice daily for 4 days and once on Day 5. Topical application of RTA 408 resulted in transdermal penetration, with low but dose-dependent plasma exposure with AUC(0-24 h) values of 3.6, 26.0, and 41.1 h ng/mL for the 0.1, 1.0, and 3.0% doses, respectively. Further, topical application of RTA 408 resulted in increased translocation of Nrf2 to the nucleus, dose-dependent mRNA induction of Nrf2 target genes (e.g. Nqo1, Srxn1, Gclc, and Gclm), and induction of the protein expression of the prototypical Nrf2 target gene Nqo1 and increased total glutathione (GSH) in normal rat skin. Immunohistochemistry demonstrated that increased staining for Nqo1 and total GSH of structures in both the epidermis and dermis was consistent with the full transdermal penetration of RTA 408. Finally, topically administered RTA 408 was well tolerated with no adverse in-life observations and normal skin histology. Thus, the data support the further development of RTA 408 for the potential treatment of skin diseases.

Dual growth factor releasing multi-functional nanofibers for wound healing

The objective of this research is to develop a dual growth factor-releasing nanoparticle-in-nanofiber system for wound healing applications. In order to mimic and promote the natural healing procedure, chitosan and poly(ethylene oxide) were electrospun into nanofibrous meshes as mimics of extracellular matrix. Vascular endothelial growth factor (VEGF) was loaded within nanofibers to promote angiogenesis in the short term. In addition, platelet-derived growth factor-BB (PDGF-BB) encapsulated poly(lactic-co-glycolic acid) nanoparticles were embedded inside nanofibers to generate a sustained release of PDGF-BB for accelerated tissue regeneration and remodeling. In vitro studies revealed that our nanofibrous composites delivered VEGF quickly and PDGF-BB in a relayed manner, supported fibroblast growth and exhibited anti-bacterial activities. A preliminary in vivo study performed on normal full thickness rat skin wound models demonstrated that nanofiber/nanoparticle scaffolds significantly accelerated the wound healing process by promoting angiogenesis, increasing re-epithelialization and controlling granulation tissue formation. For later stages of healing, evidence also showed quicker collagen deposition and earlier remodeling of the injured site to achieve a faster full regeneration of skin compared to the commercial Hydrofera Blue® wound dressing. These results suggest that our nanoparticle-in-nanofiber system could provide a promising treatment for normal and chronic wound healing.

Abstract 250: Endothelial Cell Endothelin (ET-1) Production is Enhanced with Increasing NaCl Concentration.

It has recently been demonstrated that increasing salt intake results in an accumulation of sodium within the interstitium of the skin of normal rats, which leads to an immune response that is necessary to maintain normal blood pressure. ET-1 mediates increased inflammation in several models of hypertension and is increased in response to high salt intake. However, it is unknown if ET-1 production is increased by endothelial cells in response to increasing NaCl concentration. Therefore, we hypothesized that increasing the media NaCl concentration will lead to an increase in ET-1 production by human umbilical venous endothelial cells (HUVECs). Exposure of HUVECs for 2 hours to a 40 mosm increase in NaCl results in a significant increase in ET-1 mRNA (1.65±0.14 fold increase vs. control; n=3, p&lt;0.05). This increase in Na+ concentration is similar to that observed in the interstitium of the skin of rats placed on a high NaCl diet. Furthermore, exposure to +40 mosm of mannitol, an effective osmolyte, or urea, an ineffective osmolyte, had no effect on ET-1 mRNA (1.1 ± 0.04 fold change and 1.07 ± .08 fold change vs. control, respectively; n=3, p&lt;0.05) suggesting that a direct effect of NaCl, not osmolality, is responsible for the increase in ET-1 mRNA. Moreover, +40 mosm of sodium acetate (NaAc) or choline chloride (ChCl) resulted in an increase in ET-1 mRNA very similar to that seen by increasing NaCl (1.74±0.14 (NaCl), 1.54±0.05 (NaAc), and 1.59±0.03 (ChCl) fold change vs. control, n=3, p&lt;0.05), suggesting that either the Na or Cl ion increases ET-1 expression. These data demonstrate that vascular endothelial cells express ET-1 in response to increases in NaCl concentration, and may be important in mediating the extrarenal inflammatory response to a high salt diet.

Keratinocyte expression of inflammatory mediators plays a crucial role in substance P-induced acute and chronic pain

Tibia fracture in rats followed by cast immobilization leads to nociceptive, trophic, vascular and bone-related changes similar to those seen in Complex Regional Pain Syndrome (CRPS). Substance P (SP) mediated neurogenic inflammation may be responsible for some of the signs of CRPS in humans. We therefore hypothesized that SP acting through the SP receptor (NK1) leads to the CRPS-like changes found in the rat model. In the present study, we intradermally injected rats with SP and monitored hindpaw mechanical allodynia, temperature, and thickness as well as tissue levels of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and nerve growth factor-β (NGF) for 72 h. Anti-NGF antibody was utilized to block the effects of SP-induced NGF up-regulation. Fracture rats treated with the selective NK1 receptor antagonist LY303870 prior to cast removal were assessed for BrdU, a DNA synthesis marker, incorporation in skin cells to examine cellular proliferation. Bone microarchitecture was measured using micro computed tomography (μCT). We observed that: (1) SP intraplantar injection induced mechanical allodynia, warmth and edema as well as the expression of nociceptive mediators in the hindpaw skin of normal rats, (2) LY303870 administered intraperitoneally after fracture attenuated allodynia, hindpaw unweighting, warmth, and edema, as well as cytokine and NGF expression, (3) LY303870 blocked fracture-induced epidermal thickening and BrdU incorporation after fracture, (4) anti-NGF antibody blocked SP-induced allodynia but not warmth or edema, and (5) LY303870 had no effect on bone microarchitecture. Collectively our data indicate that SP acting through NK1 receptors supports the nociceptive and vascular components of CRPS, but not the bone-related changes.

Optical delineation of human malignant melanoma using second harmonic imaging of collagen

Skin cancer incidence has increased exponentially over the last three decades. In 2008 skin cancer caused 2280 deaths in the UK, with 2067 due to malignant melanoma. Early diagnosis can prevent mortality, however, conventional treatment requires multiple procedures and increasing treatment times. Second harmonic generation (SHG) imaging could offer diagnosis and demarcation of melanoma borders non-invasively at presentation thereby short-cutting the excision biopsy stage. To test the efficacy and accuracy of SHG imaging of collagen in skin and to delineate the borders of skin cancers, unstained human melanoma biopsy sections were imaged using SHG microscopy. Comparisons with sister sections, stained with H&E or Melan-A were made for correlation of invasion borders. Fresh ex vivo normal human and rat skin was imaged through its whole thickness using SHG to demonstrate this technique is transferable to in vivo tissues. SHG imaging demonstrated detailed collagen distribution in normal skin, with total absence of SHG signal (fibrillar collagen) within the melanoma-invaded tissue. The presence or absence of signal changes dramatically at the borders of the melanoma, accurately demarcating the edges that strongly correlated with H&E and Melan-A defined borders (p<0.002). SHG imaging of ex vivo human and rat skin demonstrated collagen architecture could be imaged through the full thickness of the skin. We propose that SHG imaging could be used for diagnosis and accurate demarcation of melanoma borders on presentation and therefore potentially reduce mortality rates.

OPTICAL MEASUREMENT OF PHOTOSENSITIZER CONCENTRATION IN VIVO

Most techniques for measuring tissue concentrations of drugs are invasive, time-consuming, and often require the removal of tissue or body fluids. Optical pharmacokinetics (OP) is a minimally invasive alternative giving an immediate result. Pulses of white light are directed at the tissue of interest using a fiber optic probe. Scattered light is detected by a second fiber immediately adjacent to the first in the same probe (separation 1.7 mm). Using the photosensitizer disulfonated aluminium phthalocyanine ( AlS2Pc ), OP measurements were made in phantoms and on the mouth, stomach, colon, skin, and liver of normal rats 1 and 24 h after intravenous AlS2Pc administration. AlS2Pc concentration was determined by calculating the area under the curve (AUC) in the spectral region around the peak drug absorption or measuring the height of the peak. Spectral baseline interpolation removed the need for pre-drug, control optical measurements. OP measurements correlated well with values from alkali chemical extraction (CE) of the corresponding tissues, (R2 0.87–0.97). OP measurements in the mouth also correlated with CE of less accessible internal organs (R2 0.77–0.88). In phantoms, the lowest detectable concentration was 0.1 μg/g. In vivo, results were limited by the lower accuracy in the CE measurements but were almost certainly comparable. An incidental finding was a 12–15 nm red shifted component in the spectra observed 1 h after drug administration, suggesting partitioning of the drug in different microenvironment compartments, which could prove to be of considerable interest in future studies. In conclusion, OP shows promise for real-time measurement of concentrations of drugs with suitable absorption peaks.

Terminal arbor degeneration--a novel lesion produced by the antineoplastic agent paclitaxel.

The antineoplastic agent paclitaxel causes a dose-limiting distal, symmetrical, sensory peripheral neuropathy that is often accompanied by a neuropathic pain syndrome. In a low-dose model of paclitaxel-evoked painful peripheral neuropathy in the rat, we have shown that the drug causes degeneration of intraepidermal nerve fibers (IENFs), i.e. the fibers which give rise to the sensory afferent's terminal receptor arbor. However, we did not find any evidence for axonal degeneration in samples taken at the mid-nerve level. Here we aimed to determine whether the absence of degenerating peripheral nerve axons was due to sampling a level that was too proximal. We used electron microscopy to study the distal-most branches of the nerves innervating the hind paw glabrous skin of normal and paclitaxel-treated rats. We confirmed that we sampled at a time when IENF degeneration was prominent. Because degeneration might be easier to detect with higher paclitaxel doses, we examined a four-fold cumulative dose range (8-32 mg/kg). We found no evidence of degeneration in the superficial subepidermal axon bundles (sSAB) that are located just a few microns below the epidermal basal lamina. Specifically, for all three dose groups there was no change in the number of sSAB per millimeter of epidermal border, no change in the number of axons per sSAB and no change in the diameter of sSAB axons. We conclude that paclitaxel produces a novel type of lesion that is restricted to the afferent axon's terminal arbor; we name this lesion 'terminal arbor degeneration'.