Imperial Chemicals Industry (Alderley Edge, UK) in 1932 and maintained a closed colony. In 1969 Olac (now Har- lan Olac) obtained the stock from the Medical ...
American Journal ofPathology, Vol. 152, No. 4, April 1998 Copyright X American Society for Investigative Pathology
Animal Model A Rat Model of Spontaneous Myopathy and Malignant Hyperthermia
Luis E. Gonzalez,* Carmen V. Mel6ndez-Vasquez,t Norman A. Gregson,t and Sandra E. File* From the Psychopharmacology Research Unit* and the Depattment of Neurology,t United Medical and Dental Schools, Guy's Hospital, London, United Kingdom
Malignant hyperthermia is a main cause of death during general anesthesia, particularly in children. However, research has been hampered by the lack of a convenient animal model, the only one available being a special strain of pig. In this study, we describe spontaneous myopathy and a fatal syndrome of generalized muscle rigidity triggered by halothane in an outbred strain of rat. Histological examination of skeletal muscle reveals severe abnormalities indicating chronic underlying myopathy. The association of histological abnormalities with an acute, fatal syndrome clinically resembling malignant hyperthermia provides a strong basis for a new and extremely useful animal model to study this fatal disorder. (AmJ Patbol 1998, 152:1099-1103)
Malignant hyperthermia (MH) is a relatively rare and potentially fatal complication of general anesthesia. In North America and Europe, the overall incidence of recognized human MH reactions in children is approximately 1:15,000 anesthetics and in adults is approximately 1:50,000 anesthetics.1 Susceptible subjects have an inherited defective regulation of calcium flux in their skeletal muscles.2 Duchenne muscular dystrophy, central core disease, King-Denborough syndrome, myotonia congenita, myotonic dystrophy, and nonspecific myopathies are neuromuscular disorders commonly associated with MH.6 In some of these subjects, hypermetabolic crises with hyperpyrexia are triggered by exposure to inhalational anesthetics, depolarizing muscle-relaxants, or stress. MH is also well-documented in pigs,7e8 and fatal episodes in pigs exposed to stress have considerable economic impact. Anecdotal cases have also been reported in dogs,9 cats,10 race horses,11 cattle,12 and giraffes,13 but the only available animal model is a suscep-
tible strain of pig. Even within this strain, not all animals display the MH syndrome, and the pig is a costly and inconvenient animal for fundamental research. Here we provide for the first time the possibility of using a rat model to study the mechanisms underlying this fatal disease. We describe a syndrome with clear similarities to the clinical features of MH with a severe, chronic myopathy occurring spontaneously in an outbred strain of rat.
Materials and Methods Animals Hooded Lister rats (Harlan Olac, Bicester, UK) are a black hooded outbred strain of rat. The Medical Research Council (Mill Hill, UK) obtained the stock from Imperial Chemicals Industry (Alderley Edge, UK) in 1932 and maintained a closed colony. In 1969 Olac (now Harlan Olac) obtained the stock from the Medical Research Council. Animals were cesarean derived in the same year and again in 1972 and 1979. Randomized stock were cesarean derived into isolators in 1981. The batches of rats used here were obtained from Harlan Olac (Shaw's Farm, Blackthorn, Bicester, OX6 OTP, England). These rats have been used in behavioral studies in our laboratory for many years. The rats are transported from the breeder colonies to our unit where they are group housed and handled for 7 days before surgery. No other procedure or drug treatment is given before surgery and none of the animals showed any signs of behavioral abnormalities or ill health.
Anesthesia and Surgery The response to halothane of five batches of hooded Lister rats, between 1996 and 1997, was closely followed. Briefly, for induction, 3 to 3.5% halothane in oxygen is delivered to a plexiglass closed box (10 x 7 x 7 cm) at Supported by a grant from the Special Trustees of Guy's Hospital. Accepted for publication January 9, 1998. Address reprint requests to Professor S. E. File, Psychopharmacology Research Unit, Guy's Hospital, London SE1 9RT, UK. E-mail: s.file.@umds.ac.uk.
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Table 1. Details of Five Batches of Hooded Lister Rats Subjected to Halothane Anesthesia
Number of rats per batch 60 50 42 60 60
Cases of MH
Onset (min)
Maximum temperature (0C) during episode (rectal)
2 1 1
2-5 1 10 1 4
38.1-38.3 37.7 38.0 38.5 38.0
1*
1*
Mean (± SEM) temperature (0C) under halothane 36.5 36.2 35.7 35.3 36.7
± ± ± ± ±
0.3 0.4 0.3 0.4 0.6
Body weight range (g) of batch
280-320 300-350 280-320 300-350 250-300
Weight (g) of rat with MH
Date of surgery
290/300 320 290 310 300
November 1996 January 1997 March 1997 May 1997 July 1997
*Histological examination was performed in muscle specimens from these animals.
a flow rate of 0.5 liter/minute. For maintenance, the rats are transferred to inhalational masks in stereotaxic frames and the concentration of halothane is reduced to 2 to 2.5%. Subsequently, surgery is performed as a standard procedure for cannulation of a specific brain area.14
Histology and Immunohistochemistry Skeletal muscle specimens (triceps, deltoideus, latissimus dorsi, and quadriceps) were taken from animals immediately after death and fixed in 4% paraformaldehyde overnight at 40C. The tissue was dehydrated in a series of industrial methylated spirit (70 to 100%) with a final change into chloroform and embedded in paraffin. Ten-mm thick sections were collected on Vectabond (Vector Laboratories, Peterborough, UK) coated-slides and stained with hematoxylin and eosin and with Masson's trichrome techniques.15 For immunoperoxidase, antigen retrieval of dewaxed paraffin sections was carried out using a 600-W domestic microwave oven. Briefly, slides were placed in a vessel containing 0.01 mol/L HCI sodium citrate buffer, pH 6.0, and heated in a microwave for 15 minutes (3x 5-minute cycle). After washing, sections were blocked with a solution of 1% bovine serum albumin in phosphate-buffered saline for 1 hour, followed by incubation with 100 ml of mouse anti-ED1 antibody (Serotec, Oxford, UK) diluted to 1/200 for 1 hour at room temperature in a moist chamber. The antibody was detected using the ABC peroxidase kit (Vector Laboratories, Peterborough, UK). For immunofluorescence, muscle sections were dewaxed and pretreated with heat as described above. After washing and blocking, sections were incubated with 100 ml of anti-CD4 fluorescein isothiocyanate-conjugated mouse monoclonal antibody (Serotec) diluted to 1/1 00 for 1.5 hours at room temperature in a moist chamber. The sections were then washed and mounted in a microscope slide with a drop of Citifluor (Citifluor, London, UK). Muscle slides were examined with a Zeiss Axiophot microscope, and images were scanned and collected using a JVC Frame Capture camera (TX-F7300U) and Image Access software (Clattbrugg, Switzerland). Final image layouts were created using Adobe Photoshop and printed at a final resolution of 760 dpi.
Results MH Syndrome At least one rat from each batch died under anesthesia and showed severe and generalized muscle rigidity; the frequency of these episodes was about 1 in 50 (Table 1). The episodes were characterized by a generalized, progressive, and severe muscle rigidity occurring first in the hind limbs. Stiffness was also prominent in the longissimus dorsi, quadriceps, deltoids, extensor muscles, and the masseter muscle (jaw spasm). Mild hyperthermia, tachycardia, and tachypnea were also present. All of the animals with signs of muscle rigidity developed a full syndrome and died within 20 to 45 minutes after induction of the anesthesia. Standard resuscitation procedures such as discontinuation of halothane, cardiac massage, and artificial ventilation, which effectively rescue animals that have accidentally received anesthetic overdose, proved quite ineffective. All of our animals that were pyrexic and extremely stiff before dying became flaccid after death, ie, they did not develop rigor mortis as do normal animals. Although the temperature was not extremely elevated (Table 1), the hyperthermia was very long lasting, and rats remained at temperatures of 37 to 37.50C for at least 1 hour after death.
Histological Findings Examination by light microscopy of muscle specimens (triceps, deltoideus, latissimus dorsi, and quadriceps) taken from two animals that died under halothane showed striking abnormalities (Figure 1). Active muscle destruction with extensive fiber necrosis was evident in all of the samples examined. Focal lymphocytic and mononuclear cell infiltration was also prominent and was observed around blood vessels and in the endomysial space (Figure 1, A and B). Activated macrophages (ED1 +) and T lymphocytes (CD4+) were observed within degenerated fibers undergoing phagocytosis (Figure 1, C and D). Several clusters of basophilic regenerating fibers in association with vesicular nuclei were also present (Figure 1 E). Other pathological changes, such as fiber splitting, hyaline and atrophic fibers, central nuclei, and fibrosis, were also found (Figure 1, F to H). The nerves within the muscles appeared normal, and there
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C
Figure 1. Paraffin sections from skeletal muLscle of rats that died under exposure to halothane. A and B: H&E stain showing extensive endomysial mononuclear cell infiltration with invasion of necrotic fibers. C: Immunoperoxidase staining of activated macrophages within a necrotic fiber identified with mouse monoclonal antibody ED1. Only some of the cells within the muscle were EDI+. The section was counterstained with Harris' hematoxylin. D: Immunofluorescence staining of endomysial infiltration of T lymphocytes (arrowheads). The section was incubated with mouse monoclonal anti-CD4+ fluLorescein isothiocyanate-conjuLgated antibody. E: Zones of clustered regenerating fibers identified by their small caliber and basophilic cytoplasm with prominent sarcolemmal nuLclei, section stained with H&E. F: Large and rounded hyaline fiber (solid arrowhead) is seen in this section stained with Masson's trichrome. There is also a split fiber (empty arrowhead) and a marked variability in the fiber size. Inflammatory cells are seen around a thickened blood vessel. G: The fiber in the middle of this section, stained with Masson's trichrome, contains a central nucleus. Enlarged and small regenerating fibers are present and there is a moderate inflammatory infiltrate in between the fibers. H: This section, stained with Masson's trichrome, shows regenerative myoblasts in many muscle fibers undergoing phagocytosis. Scale bars, 200 mm (A); 50 mm (B, E, and F); 25 mm (C, G, and H); 20 mm (D).
signs of nerve inflammation, demyelination, or degeneration. These histological features indicate the occurrence of an active myopathic disorder in the animals susceptible to the hyperthermic syndrome triggered by halothane. Interestingly, the histological examination of identical muscle specimens from hooded Lister rats that did not develop the MH syndrome on exposure to halothane also demonstrate some small abnormalities such as cell infil-
were no
tration and fibrosis (Figure 2, A and B). However, these changes were less common and less severe in these animals. Skeletal muscle from Lewis rats used as a strain control did not show any pathological change. Microscopic examination of coronal brain sections within the hypothalamic areas (1.3 to 4.3 mm posterior to bregma) did not reveal any anatomical abnormality in these animals. Similarly, macroscopic examination of thoracic and abdominal organs did not reveal any gross abnormality.
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These results suggest that the pathological changes were restricted to muscle tissue.
Discussion The finding that animals were stiff and pyrexic before death but then became flaccid indicates that a hypermetabolic process triggered by the anesthetic may have produced biochemical alterations in the muscle. As the fever is a result, not a cause, of the hypermetabolism in the muscles during MH crises, it tends to be of relatively late onset. There have even been reports of an MH syndrome without hyperthermia.16 It is possible that the mild hyperthermia observed in our rats was because this fulminant syndrome developed so rapidly that the body temperature did not reach an extreme level and also because small animals lose body heat rapidly. The striking pathological changes found in the muscles of the rats that developed the MH syndrome are those that characterize chronic disorders, and it is therefore unlikely that they resulted from acute damage to the muscle during the MH episode. Muscle biopsies from survivors of MH crises did not show the type of changes that we report.17-20 We believe that they may represent a subclinical, chronic, myopathic disorder that renders these animals more susceptible to MH. It was clear from our results that the pathological changes are restricted to muscle tissue. The association of malignant hyperthermia with a number of neuromuscular disorders is well documented in humans,6 but there are no previous reports of a similar condition in rats. The most important clinical goal in MH is to detect, before exposure to anesthesia, abnormalities that may predispose an individual to an MH episode, and thus identification of all MH mutations is necessary. Currently, an in vitro contracture test is widely used to screen MH susceptibility,21 but this was not technically feasible in our laboratories. However, this test is not very specific22 and false negatives have been reported.23 The causative role of the mutations identified for MH is unclear and genetic diagnosis is not yet possible.2 The only clinical antidote is dantrolene, which has to be administered
immediately as any symptoms appear. This has reduced the mortality rates, but considerable morbidity remains from neurological or kidney damage. Other aspects of this syndrome also need additional investigation, eg, its clinical variability,24 the discordance between phenotype and genotype,25 the abnormal regulation of calcium release induced by inositol 1,4,5-triphosphate,26 and the potential relationship of MH to hypermetabolic crises triggered by stress27 and drugs, such as neuroleptics,28'29 ecstasy,30 and cocaine.31 There is world-wide interest in MH and there have been over 1000 research publications in the last 10 years. The availability of a phenotype of MH in the rat will greatly facilitate genetic and biochemical studies of this disorder. We strongly believe that the association that we have found of histological abnormalities indicative of a chronic myopathic disorder and an acute and fatal syndrome of generalized rigidity triggered by halothane provides strong evidence for a model of MH.
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724-727 31. Sato N, Brum JM, Mitsumoto H, DeBoer GE: Effect of cocaine on the contracture response to 1% halothane in patients undergoing diagnostic muscle biopsy for malignant hyperthermia. Can J Anaesth 1995, 42:158-162
