Tracey A. Hamilton

Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice†

Nerve‐evoked contractions were studied in vitro in phrenic nerve–hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE−/−) mice and their wild‐type littermates (AChE+/+). The AChE−/− mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE−/− mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild‐type animals. In AChE−/− preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5‐bis(4‐allyldimethyl‐ammoniumphenyl)pentane‐3‐one (BW 284C51). The ability of diaphragm muscles from AChE−/− mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms—including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region—were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors. Muscle Nerve 30: 317–327, 2004

Sulfur Mustard-Induced Apoptosis in Hairless Guinea Pig Skin

The present study was aimed to examine whether apoptosis is involved in the pathogenesis of sulfur mustard (SM)-induced basal cell death. Skin sites of the hairless guinea pig exposed to SM vapor for 8 minutes were harvested at 3, 6, 12, 24, and 48 hours postexposure. Immunohistochemical detection of basal cell apoptosis was performed using the ApopTag in situ apoptosis labeling kit. Only occasional apoptotic basal cells (BC) were observed in nonexposed and perilesional control sites. At lesional sites, apoptosis of BC was not detected at 3 hours postexposure. However, at 6 hours and 12 hours postexposure, 18% and 59% of BC were apoptotic, respectively. At 24 and 48 hours postexposure, individual apoptotic basal cells were not clearly recognizable due to necrosis. At the ultrastructural level, degenerating BC exhibited typical apoptotic morphology including nuclear condensation and chromatin margination. The results suggest that apoptotic cell death is a cytotoxic mechanism with the number of BC undergoing apoptosis significantly increasing from 6 to 12 hours postexposure. In addition, because necrosis is preferential at 24 hours postexposure, we believe that SM-induced cell death involves early apoptosis and late necrosis, which temporally overlap to produce a single cell death pathway along an apoptotic-necrotic continuum.

Immunohistochemical studies of basement membrane proteins and proliferation and apoptosis markers in sulfur mustard induced cutaneous lesions in weanling pigs

Sulfur mustard (2,2-dichlorodiethyl sulfide, HD) is a chemical warfare agent that is a threat to both troops and civilians. The focus of HD research has been on intracellular adduct formation leading to apoptosis and/or necrosis in cutaneous lesions. However, there is work which suggests that HD may have a more direct effect on the basement membrane zone. Immunohistochemical staining to desmosomal proteins, cellular fibronectin, laminin 1, laminin 5, collagen IV, collagen VII, p53, Bcl-2, and PCNA was performed on weanling pig skin exposed to vesicating doses of HD, GB3, an antibody to laminin 5, showed a progressive decrease with loss of expression during the time period of clinical vesiculation. The other basement membrane proteins showed no change or inconsistent changes. PCNA, and p53 staining increased in the overlying epidermis in areas of vesiculation without significant necrosis. Bcl-2 positive cells were decreased or absent after exposure. This study implicates laminin 5 as the main basement membrane protein affected acutely by HD exposure. The patterns of staining of PCNA, Bcl-2, and p53 within the epidermis suggest that apoptosis and cellular necrosis both may play a role in cell death secondary to HD.

Microvesicating effects of sulfur mustard on an in vitro human skin model

Bis-(beta-chloroethyl) sulfide (SM) is a potent skin vesicant previously used for chemical warfare. Progress in determination of the mechanistic basis of SM pathology, and development of prophylactic and/or therapeutic countermeasures to SM exposure has been hampered by lack of physiologically relevant models of human skin. The current work evaluated a newly developed tissue engineered full-thickness human skin model in a completely in vitro approach to investigation of SM-induced dermal pathology. The model was first characterized with regard to overall morphology, lipid composition, basement membrane (BM) composition and ultrastructural features that are important targets of SM pathologic activity. Well-developed BM ultrastructural features were observed at the dermal-epidermal junction (DEJ), thus demonstrating successful resolution of a primary deficiency of models previously evaluated for SM studies. Studies were then conducted to evaluate histopathological effects of SM on the model. Good replication of in vivo effects was observed, including apoptosis of basal keratinocytes (KC) and microblister formation at the DEJ. Tissue engineered skin models with well-developed basement membrane structures thus appear to be useful tools for in vitro mechanistic studies of SM vesicant activity and development of preventive/therapeutic approaches for SM pathology.

Depth of morphologic skin damage and viability after one, two, and three passes of a high-energy, short-pulse CO 2 laser (Tru-Pulse) in pig skin

BACKGROUND CO2 laser energy is absorbed by water, which is present in all tissue. The depth of penetration of CO2 lasers is narrow with minimal reflection, scatter, or transmission. However, thermal damage has limited the usefulness of conventional, continuous-wave CO2 lasers for debridement as demonstrated by wound healing studies. The development of high-energy CO2 lasers, with pulse durations that are less than the thermal relaxation time of tissue, have made vaporization of skin for resurfacing and wound debridement possible because of the decreased risk of thermal damage. OBJECTIVE This study was performed to evaluate thermal damage produced by a CO2 laser. METHODS Routine histopathologic examination and nitroblue-tetrazolium chloride (NBTC) staining were used to evaluate the depth of tissue damage and viability in weanling pig skin after one, two, and three passes of the laser. RESULTS At a pulse energy of 300 mJ, with a pulse duration of 60 microseconds, one pass of the laser produced vaporization of the epidermis with minimal thermal damage. Two passes produced areas of denatured collagen with loss of viable cells in the superficial papillary dermis. Three passes extended the damage into the papillary dermis. CONCLUSION Hyalinization of collagen appears to correspond well with the level of thermal damage as measured by NBTC staining. Our findings suggest that the energy necessary to vaporize the dermis may be greater than that needed to vaporize epidermis.

Immunolocalization of MAP-2 in Routinely Formalin-Fixed, Paraffin-Embedded Guinea Pig Brain Sections Using Microwave Irradiation: A Comparison of Different Combinations of Antibody Clones and Antigen Retrieval Buffer Solutions

The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity.

Sensitivity of cross-reacting antihuman antibodies in formalin-fixed porcine skin: including antibodies to proliferation antigens and cytokeratins with specificity in the skin

Although no animal is a perfect skin model for the study of toxicological and therapeutic agents, structurally the pig may be superior to even non-human primates. Because our work involves effects of toxicological and therapeutic agents on the skin, we wanted to identify stains which may prove useful as well as determine cross-reactivity of some newer antihuman antibodies. We performed a battery of formalin-fixed skin from weanling pigs and minipigs. The battery of antibodies included LCA, CD3, OPD-4, CD34, UCHL-1, L-26, KP-1, MAC-387, Factor XIIIa, Leu-7, S-100 protein, HMB-45, GFAP, synaptophysin, neurofilament protein, ubiquitin, vimentin, type IV collagen, laminin, fibronectin, Factor VIII related antigen, Desmin-M, smooth muscle actin, cytokeratin 7, cytokeratin 20, AEI/AE3, CAM 5.2, EMA, GCDFP, Ki-67, and PCNA. Immunohistochemical stains for CD3, Leu-7, S-100 protein, type IV collagen, laminin, Factor VIII related antigen, GFAP, synaptophysin, neurofilament protein, ubiquitin, smooth muscle actin, vimentin, Desmin-M, cytokeratin 7, cytokeratin 20, AE1/AE3, CAM 5.2, Ki-67 and PCNA showed consistent cross-reactivity. In formalin-fixed tissue, only antibodies to lymphoreticular cells showed poor cross-reactivity. A high percentage of the remaining antibodies did show good cross-reactivity but with some interesting similarities and differences in specificity.