JAC Advance Access originally published online on November 30, 2005
Journal of Antimicrobial Chemotherapy 2006 57(2):294-300; doi:10.1093/jac/dki430
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Pathological changes in the brains of rabbits experimentally infected with Angiostrongylus cantonensis after albendazole treatment: histopathological and magnetic resonance imaging studies
1 Department of Parasitology, College of Medicine, Chang-Gung University, Kueisan, Taoyuan 333, Taiwan; 2 Department of Pathology, Chang-Gung Memorial Hospital, Chang-Gung Children Hospital at Linkou and Chang-Gung University, Kueisan, Taoyuan 333, Taiwan; 3 Department of Diagnostic Radiology, Chang-Gung Memorial Hospital at Linkou, College of Medicine, Chang-Gung University, Kueisan, Taoyuan 333, Taiwan; 4 College of Arts and Sciences, Case Western Reserve University, Cleveland, OH, USA
* Corresponding author. Tel: +886-3-328-1200 ext. 2575; Fax: +886-3-397-1936; E-mail: ylw0518{at}adm.cgmh.org.tw
Received 14 January 2005; returned 15 May 2005; revised 1 July 2005; accepted 24 October 2005
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Objectives: To determine the effects of albendazole on rabbits infected with larvae of Angiostrongylus cantonensis by histopathological and magnetic resonance imaging (MRI) techniques.
Methods: Male rabbits were infected with 400 A. cantonensis larvae and treated with albendazole (5 mg/kg/day) for 2–14 days on day 5, 10, 15 or 20 post-infection.
Results: Although there were pathological changes in the brains, MRI revealed unremarkable findings in the untreated group. However, the treated rabbits exhibited eosinophilic meningitis, choroid plexus inflammation, meningeal congestion, encephalitis, perivascular cuffing and meningitis, and were also found to have abnormal signal intensities on brain MR images in the 20 day post-infection treated group.
Conclusions: Pathological changes in the brains of the treated rabbits are more severe than those without albendazole treatment, suggesting that the drug may not be very suitable for the treatment of cerebral angiostrongyliasis.
Keywords: A. cantonensis , MRI , pathology , histopathology , angiostrongyliasis , anthelmintics
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Introduction |
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Angiostrongylus cantonensis, the rat lungworm, is the most common causative agent of human eosinophilic meningitis or eosinophilic meningoencephalitis. Humans acquire the infection by ingesting contaminated snails of Achatina and Pila, slugs, planaria or freshwater prawns. The third-stage larvae then migrate to the brain and develop into the fifth stage.1 Patients usually present an insidious or sudden onset of excruciating headache, neck stiffness, nausea, vomiting and paraesthesia. Clinical manifestations such as fever, cranial nerve palsies, seizures, paralysis and lethargy are less common. Most patients with cerebral angiostrongyliasis have a self-limited course and recover without sequelae. Management usually consists of symptomatic and supportive treatments. Removal of CSF by spinal puncture is applied to patients with severe headache. Corticosteroids are administered to patients with severe inflammatory reactions. Surgical operation is required only in those with ocular involvement.2
Albendazole has been reported to be the drug of choice against larvae of A. cantonensis. In addition, anthelmintics such as thiabendazole, 1-tetramisole, mebendazole, avermectin B1a, ivermectin, flubendazole and milbemycin have also been evaluated to be effective in the treatment of this disease.3,4 Although the chemotherapeutic values of these drugs have been determined by the conventional techniques, no in situ information on the mechanisms of their actions is available. Since these larvicidal agents have been considered to be able to exacerbate the inflammatory brain lesions,5–8 there is controversy regarding the administration of chemotherapy.
Magnetic resonance imaging (MRI) has been suggested to be a non-invasive technique in localizing and characterizing lesions during the acute phase of angiostrongyliasis due to A. cantonensis.9 This technique is a well-established imaging modality for clinical evaluation of lesions in the CNS and its application in the field of parasitology is fruitful. The potential of MRI in the diagnosis of cysticercosis and hydatidosis has been demonstrated.10,11 In comparison with computerized tomography (CT), MRI provides higher tissue contrast and superior sensitivity in the detection of lesions. CT is extremely sensitive and specific in delineating calcification, whereas MRI is sensitive in showing tissue change and oedema. These two techniques have been recommended as complementary tools in the diagnosis of neurocysticercosis.12 Recently, various classifications of the disease have been reported in the United States13 and France.14 Moreover, MRI has been used in the diagnosis of lesions due to Toxocara canis in the spinal cord15 as well as in the post-therapeutic surveillance on African trypanosomiasis.16
In previous studies on the effectiveness of anthelmintics against the larvae of A. cantonensis, only the recovery of the host in response to different dosages was emphasized whereas the pathological changes in the brain of the host were rarely assessed.3,4 In order to determine the effects of albendazole on rabbits infected with A. cantonensis larvae, we employed histopathology and MRI to study the lesions in animal brains after drug treatment at different stages.
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Parasite and laboratory animals
A. cantonensis has been maintained in our laboratory since 1980. It cycles through Biomphalaria glabrata snails and Sprague-Dawley rats.17 Male New Zealand white rabbits (1.5 kg) were purchased from the Laboratory Animal Center, College of Medicine, National Taiwan University at Taipei and National Veterinary Research Institute, Chunan at Hsinchu. These animals were then kept at the Laboratory Animal Center of Chang-Gung University until they attained a body weight of 2.5–3.0 kg. All of the procedures involving animals and their care in this study were approved by the Institutional Animal Care and Use Committee of Chang-Gung University in accordance with institutional guidelines for animal experiments.
Experimental infection
Third-stage larvae of A. cantonensis were isolated from the infected B. glabrata snails. After being anaesthetized with 0.6 mL of ketamine (50 mg/mL) in 2% Rompum solution (0.6 mL), each rabbit was infected with 400 larvae by stomach intubation. The infected animals were separately reared in stainless steel cages and provided with food and drinking water ad libitum.
Drug administration
After experimental infection, albendazole (5 mg/kg/day) was administered to 28 rabbits. These rabbits were divided into four groups and received the treatment on day 5, 10, 15 or 20 post-infection (PI). In each group, the drug was orally administered to each animal for 2, 4, 6, 8, 10, 12 or 14 days. The effects of albendazole on the brains of the infected rabbits at different stages PI were evaluated by MRI and histopathology. In addition, one control group (comprised eight untreated rabbits) was also examined.
MRI
MR scans were performed on a 1.5 Tesla whole-body scanner (Magnetom Vision; Siemens Medical Systems, Erlangen, Germany) with a field gradient strength of 25 mT/m using a standard quadrature head coil. The rabbits were anaesthetized with ketamine (1.5 mL) in 2% Rompum solution (1.5 mL). Coronal T1-weighted images through the entire brain were obtained using a spin-echo sequence with a time for repetition/time for echo (TR/TE) of 464/3.5 ms, a slice-thickness of 1.5 mm, distance factor of 0.1, 80 mm field of view and a 256 x 256 imaging matrix. Coronal T2-weighted images through the entire brain were obtained using a fast spin-echo sequence with a TR/TE of 2136/119 ms, a slice-thickness of 2.0 mm, distance factor of 0.1, 90 mm field of view and a 240 x 256 imaging matrix.
Ten radiofrequency (RF) excitations for T1-weighted images and 15 RF excitations for T2-weighted images were employed. The RF excitations were summed for signal averaging to increase the signal-to-noise ratio (SNR) (SNR is determined by signal intensity of tissue/standard deviation of noise measured outside the body). The acquisition time for each rabbit at any stage was 19 min and 51 s for T1-weighted images and 8 min and 34 s for T2-weighted images. Ten to eleven excitations were summed for signal averaging. The anatomical regions on the MR images were labelled according to Shek et al.18 After obtaining pre-enhanced T1- and T2-weighted MR images, post-enhanced T1-weighted MR images were obtained following intravenous administration of gadolinium-diethylenetriaminepentaacetate (Gd-DTPA; Magnevist, Schering AG, Pharmaceutical Division, Berlin, Germany) with a dosage of 0.1 mmol/kg.
Two radiologists with more than 15 years of professional experience independently interpreted the MR images without the knowledge of pathological changes in the rabbit brain. To increase the reliability and accuracy of the interpretations, brain MR images of non-infected rabbits were reviewed before image analysis. Findings of abnormal signal intensities and abnormal enhancement at different anatomical locations on the MR images were recorded. A consensus was reached when there were discrepancies between the MRI findings.
Histopathological examination
Immediately after performing the MRI, the rabbits were sacrificed with an intravenous overdose of pentobarbital. The brains were removed from the cranial cavity and fixed in 10% formalin. After fixation for 3 weeks, the brains were embedded in paraffin, sliced in 4–5 mm thick coronal sections and stained with haematoxylin and eosin. The stained sections were then examined under a light microscope.
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Histopathological findings
In the untreated group, each of the eight infected rabbits was found to have variable pathological changes in the brain tissue (Table 1). Seven rabbits (87.5%) showed infiltrates of eosinophils in the meninges of cerebrum. This change predominated in the dorsal part. Congestion was also noted in the meninges in two cases (25.0%). Infiltrates of lymphocytes and eosinophils in the brain parenchyma were found in four cases (50.0%). Two cases (25.0%) showed focal necrosis in the brain tissue. Perivascular cuffing of lymphocytes was noted in the brain tissue of one rabbit (12.5%) and inflammation in the mammillary body in one case (12.5%). In six rabbits (75.0%), the choroid plexus in the lateral ventricles showed infiltrates of lymphocytes and eosinophils. These lesions were <200 µm in diameter and 50 x 100 µm2 in dimension (Figure 1). Moreover, fifth-stage larvae of A. cantonensis surrounded by inflammatory cells were found in three rabbits (Figure 1a–c).
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Except for one rabbit treated with albendazole for 14 days in the 15 day PI group, one or more histopathological changes were observed in the brain tissue of the treated animals. Infiltrates of eosinophils in the meninges were noted in 22 cases (78.6%) and congestion in the meninges in five cases (17.9%). In 11 rabbits (39.3%), infiltrates of lymphocytes and eosinophils were found in the choroid plexus of the lateral ventricles. Encephalitis was noted in four cases (14.3%) and perivascular cuffing of lymphocytes in three cases (10.7%) (Table 2). Larvae of A. cantonensis surrounded by inflammatory cells were found in two rabbits (Figure 2). Eosinophilic meningitis was the most common change and was found in 22 rabbits. This change was absent in only four rabbits treated for 2–8 days in the 5 day PI group, one rabbit treated for 2 days in the 10 day PI group, and the rabbit that was free from any pathological changes (Table 2). Although brain necrosis was not found in the treated groups, the lesions were larger than those revealed in the untreated rabbits (Figure 2).
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MRI findings
Of the untreated eight rabbits, no abnormal findings were observed on brain MR images. Although A. cantonensis larvae were found in the brain sections of the rabbits infected for 22 and 26 days, there were no abnormal signals in the same anatomic regions (Figure 3).
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Abnormal findings on MR images were observed in the 20 day PI group. Hyperintensities were found on T2-weighted brain MR images of the rabbits treated for 2, 6, 8 and 12 days (Figure 4). The pre-enhanced T1-weighted MR images revealed hypointensities near the left and right hippocampus, left and right lateral ventricles, right tractus, and left capsula interna (Figure 4a, d, g and j). Post-enhanced T1-weighted MR images after intravenous administration of gadolinium showed hypointensities without enhancement in the same areas (Figure 4b, e, h and k). T2-weighted MR images showed hyperintensities near the left and right hippocampus, left and right lateral ventricles, right dentatus gyrus, and right tractus geniculocalcarine (Figure 4c, f, i and l).
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Previous reports on pathological changes in the CNS caused by A. cantonensis are mainly based on post-mortem examination. Rosen et al.5 found seven larvae of A. cantonensis in the brain and meninges of a mental patient in Hawaii. Sonakul reported four fatal cases of human angiostrongyliasis. In addition to the fourth- or fifth-stage larvae found in the meninges, brain tissues, blood vessels and perivascular spaces, tracks and microcavities were found throughout the brain and spinal cord. Moreover, infiltration of the meninges and intracerebral vessels by lymphocytes, plasma cells and eosinophils was observed.19 Witoonpanich et al.20 reported multiple tracks and cavities with the presence of A. cantonensis in the brain and spinal cord in a fatal case of eosinophilic myelomeningoencephalitis after ingestion of Pila snails. Cooke-Yarborough et al. reported the finding of numerous nematodes identified as A. cantonensis in the vessels of the lungs, brain and spinal cord in an 11-month-old infant who died of angiostrongyliasis. The presence of adult nematodes in the lungs and in the brain and spinal cord could have been attributed to pulmonary abscesses and eosinophilic meningitis.21
There are a number of reports on the application of MRI in the diagnosis of angiostrongyliasis caused by A. cantonensis. Clouston et al.9 reported radicular and cerebral parenchymal involvements in brain MR images of a patient with eosinophilic meningitis. Ogawa et al.22 demonstrated multiple small hyperintense areas on Gd-enhanced T1-weighted MR images of a 17-year-old girl with eosinophilic meningoencephalitis. Kanpittaya et al.23 reported brain MR images of six patients with eosinophilic meningoencephalitis featuring prominence of Virchow–Robin spaces, subcortical enhanced lesions and abnormally high T2 signal lesions in the periventricular regions. Based on the brain MR images of 13 patients, Tsai et al.24 showed high signal intensities in the globus pallidus and cerebral peduncle on T1-weighted images, leptomeningeal enhancement, ventriculomegaly and punctate areas of abnormal enhancement within the cerebral and cerebellar hemispheres on Gd-enhanced T1-weighted images, and hyperintense signals on T2-weighted images.
In the present study, the infected rabbits without treatment revealed pathological changes in their brain. The larva or choroids plexus inflammation infiltrated with eosinophils and giant cells were detected in the rabbit brain on days 21–26 after infection. The lesions were <200 µm in diameter and 50 x 100 µm2 in dimension (Figure 1). However, the brain MR images of these rabbits showed no abnormal findings. These negative results may be attributed to the limitation of spatial resolution in the low field strength of the MR scanner combined with the small size of the larvae. The length of A. cantonensis larvae has been reported to be <0.5 mm in the second moult on day 21 PI and <20 mm at the end of the fourth moult on day 29 PI.25 However, we have revealed positive findings on brain MR images near the left lateral ventricle and hippocampus in a rabbit on day 28 PI.26
Although there are controversies on the necessity of chemotherapy against A. cantonensis infection, a number of anthelmintics have been reported to be effective. Jindrak and Alicata27 found that two doses of levamisole (80 mg/kg) administered at 5 and 6, or 10 and 11, or 22 and 23 days PI may kill all A. cantonensis in the cranial cavity of rats. Ishii et al.28 administered avermectin B1a orally to rats infected with A. cantonesis larvae at a dose of 1 mg/kg at 3 days PI and found 60% reduction in worm recovery. On days 5–9 PI, oral administration of 12.5 mg/kg/day of levamisole or mebendazole may completely eradicate A. cantonensis larvae in rats.29 A single oral dose of 100 mg/kg of mebendazole on days 6, 7 or 10 PI may be completely effective in treating rats infected with the A. cantonensis larvae.30 Oral administration of flubendazole or mebendazole at 10 mg/kg/day for 5–7 days PI may eliminate 93–97% of A. cantonensis larvae in mice and 100% of the larvae in rats. However, administering milbemycin D at 10 mg/kg on day 10 PI may be partially effective against A. cantonensis larvae in mice, whereas a dosage of 10 mg/kg/day for 10 consecutive days (days 61–70 PI) may have only minimal effect against adult A. cantonensis in rats.6
Hwang and Chen4 reported that albendazole was effective in the treatment of A. cantonensis in mice when administered within 15 days PI. This study revealed that the rabbits with eosinophilic meningitis could be effectively treated by administering the drug in the early stages of infection (Table 2). In contrast, pathological changes in the brain were found in nearly all rabbits treated at later stages PI. Hyperintense regions were found on T2-weighted brain MR images of the rabbits in the 20 day PI group treated for 2, 6, 8 and 12 days. Based on the results of MRI and dimensions of the lesions, the pathological changes in the brains of the treated rabbits are more severe than those without albendazole treatment. It may not be very appropriate to use albendazole for treating cerebral angiostrongyliasis.
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Acknowledgements |
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We thank Mr Shao-Wai Lee, Department of Diagnostic Radiology, Chang-Gung Memorial Hospital, for his help in acquiring and analysing the magnetic resonance images, and Miss Chia-Lin Yang, Miss Jan-Mei Lo, Miss Chia-Chen Ko, Miss Shin-Yi Huang and Miss Mu-Jung Chen for their technical assistance. This study was supported in part by grants from the National Health Research Institute, Republic of China (NHRI-EX91-8908EL), the National Science Council (NSC90-2320-B182-034), and the Chang-Gung Medical Research Program (CMRP970).
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