Mutsuhiko Mizobuchi

Bell palsy and herpes simplex virus : identification of viral DNA in endoneurial fluid and muscle

Bell palsy is the most common cause of facial paralysis worldwide; it has an incidence of 20 to 30 per 100 000 persons [1]. Although the second most frequent cause of facial paralysis, the Ramsay-Hunt syndrome, is known to be caused by reactivated varicella-zoster virus [2], the etiologic agent responsible for Bell palsy has not been identified. Many events, such as viral infection [1, 3, 4], ischemia [5], and autoimmune reaction [6], have been proposed as causes of Bell palsy. Viral infection is thought to be the most likely cause [7]. However, it is rare to find a diagnostic fourfold increase in specific viral antibody titer in the acute and convalescent serum specimens of patients with Bell palsy [3, 4, 7]. Postmortem histopathologic studies of the facial nerve suggest viral neuritis [8], but electron microscopic studies have failed to detect specific viral particles in the facial nerve [9]. Because the etiologic agent of Bell palsy is unknown, treatment of this condition is empiric, varying from observation alone to the use of steroids, surgical decompression, and antiviral agents. We analyzed the viral genomes of herpes simplex virus type 1 (HSV-1), varicella-zoster virus, and Epstein-Barr virus using polymerase chain reaction (PCR) on facial nerve endoneurial fluid specimens and specimens of posterior auricular muscle innervated by the facial nerve. Methods Patients and Specimens During a 4-year period, 14 of 170 patients with Bell palsy and 9 of 51 patients with the Ramsay-Hunt syndrome had decompression surgery 12 to 87 days after the onset of facial palsy. None had benefited from medical management. All patients and controls gave informed consent. Two types of clinical specimens were collected intraoperatively: endoneurial fluid from the facial nerve and tissue from the posterior auricular muscle. A piece of auricular muscle was resected after skin incision, and we obtained endoneurial fluid by absorbing it with a small, sterilized surgical sponge held at the mastoid segment immediately after the epineural sheath was incised. We stored both specimens immediately at 80 C and continued to store them at that temperature until PCR analysis was done. Control specimens of endoneurial fluid and posterior auricular muscle were collected during decompression surgery from four patients with temporal bone fracture or bacterial infection concomitant with otitis media. Posterior auricular muscle specimens were obtained during tympanoplasty from five patients with chronic otitis media who did not have facial paralysis. As an additional control, a piece of neural tissue was obtained from each of three patients with parotid tumors or facial neuroma whose facial nerves had already been affected (Table 1). Table 1. Clinical Data and Polymerase Chain Reaction Results in Patients with Bell Palsy, Patients with the Ramsay-Hunt Syndrome, and Controls* Polymerase Chain Reaction To amplify and identify the HSV, varicella-zoster virus, and Epstein-Barr virus genomes, five sets of virus-specific primers and internal oligonucleotide probes were synthesized for PCR and Southern blot analysis. Primer set 1 was prepared for amplification of the HSV-1 genome, which is located on the US6 gene [10]. Primer set 2 was designed to amplify both HSV-1 and HSV-2. A sense primer (5-CCACCGAGCGGCAGGTGATC-3) and an antisense primer (5-GCCGACCGCCTGCTCGTGCT-3) are located on the UL44-45 gene [11]. Using primer set 2, we discriminated between HSV-1 (578 base pairs) and HSV-2 (621 base pairs) on the basis of size. Nucleotide sequences of the HSV specific internal probe are 5-GAGGCGATCGAGTGGGT-3. Primer sets 3 and 4 were prepared for varicella-zoster virus amplification (genes 29 and 62, respectively) [12], and primer set 5 was prepared for Epstein-Barr virus amplification (latent cycle gene) [13]. The sensitivities of the primer sets were assessed by making serial 10-fold dilutions of each purified DNA sample. The limits of detection for primer sets 1, 2, 3, 4, and 5 were about 10, 100, 10, 100, and 10 femtograms, respectively. Samples of endoneurial fluid (about 10 L), posterior auricular muscle (2 mg to 5 mg), and nerve tissue (0.5 mg to 1 mg) were completely digested with proteinase K. Polymerase chain reaction amplification and subsequent hybridization with Southern blot analysis were done as described previously [14]. Rigid precautions against contamination in the sample processing included the use of water controls replacing the DNA samples in all amplifications. Serum Antibody Titers We examined serum antibody titers by using the complement fixation test for HSV-1 and varicella-zoster virus and by using fluorescent antibody methods for Epstein-Barr virus 8 to 38 days after the onset of facial paralysis. Results We amplified HSV-1-specific DNA fragments from the US6 and UL44-45 genes in both endoneurial fluid and posterior auricular muscle specimens obtained from patients with Bell palsy. The PCR-amplified products of the US6 gene were detected by Southern blot analysis in 10 of the 13 fluid specimens (77%) and 8 of the 14 muscle specimens (57%); the products of the UL44-45 gene were detected in 4 of the 13 fluid specimens (31%) and 7 of the 14 muscle specimens (50%). Neither varicella-zoster virus nor Epstein-Barr virus was detected in the same clinical specimens (Figure 1, top; (Table 1). We did not detect HSV-1 DNA in either the fluid or the muscle specimens of three patients with Bell palsy [patients 9, 12, and 13]. The PCR-amplified DNA fragments of the US6 gene from two patients with Bell palsy (patients 6 and 7) were sequenced directly after asymmetric PCR was done as described previously [14]. The nucleotide sequences of the amplified products (221 base pairs) were identical to those of the HSV-1 genome submitted to the GenBank (Mountain View, California [data bank with genetic information]) with accession numbers J02217 and K02372. Figure 1. Amplification of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) genomes from clinical samples. top bottom middle We detected varicella-zoster virus DNA from gene 29 or gene 62 in specimens obtained from patients with the Ramsay-Hunt syndrome only (Figure 1, middle; Table 1). Gene 29 was detected in 8 of the 9 patients (89%), and gene 62 was detected in 6 of the 9 patients (67%). On the other hand, we could not amplify HSV-1, varicella-zoster virus, or Epstein-Barr virus DNA from any specimens obtained from the other controls (Figure 1, bottom; Table 1). Serum antibody titer to HSV-1 was positive in 12 of 13 patients (92%) with Bell palsy, in 4 of 9 patients (44%) with the Ramsay-Hunt syndrome, and in 5 of 9 controls (56%) (Table 1). The prevalence of HSV-1 antibody in patients with Bell palsy was significantly higher than that in controls (P < 0.05, Fisher exact test). However, antibody titers to HSV-1 in patients with Bell palsy were not significantly higher than those of controls, as previously reported [3, 4]. Discussion We found HSV-1 DNA in 11 of 14 patients (79%) with Bell palsy, and we found varicella-zoster virus DNA in 8 of the 9 patients (89%) with the Ramsay-Hunt syndrome. The identification of viral DNA may not always be definitive evidence that a particular agent causes a disease process, because PCR can amplify viral DNA regardless of whether the virus is in the infective, lytic, or latent state. The presence of latent HSV-1 and varicella-zoster virus genomes has also been shown by PCR in the geniculate ganglion of human facial nerves at autopsy [15-17]. However, HSV-1 and varicella-zoster virus usually remain dormant in ganglia and would probably not be detected in the endoneurial fluid or auricular muscle unless they were reactivated. This hypothesis was supported by our inability to detect either HSV-1 or varicella-zoster DNA in controls. Cranial nerve surgery often reactivates latent HSV, causing labial and facial herpetic lesions 48 to 72 hours after surgery [18]. However, because we obtained specimens within 2 hours of beginning decompression surgery, reactivation of the virus in the muscle or fluid was probably not induced by surgery. If this surgery did reactivate latent HSV-1, viral DNA should also have been detected in patients with the Ramsay-Hunt syndrome and in other controls who were seropositive for HSV-1. Triggers known to be associated with Bell palsy are also known to reactivate HSV. Preceding stress, such as upper respiratory tract infection, fever, dental extraction, menstruation, or exposure to cold might reactivate latent HSV-1 in the geniculate ganglion. After the virus reactivates, it destroys ganglion cells and spreads into the endoneurial fluid. The virus also infects Schwann cells, leading to demyelinization and inflammation of the facial nerve [19]. This inflammatory response has been shown by gadolinium-enhanced magnetic resonance imaging in patients with Bell palsy and in patients with the Ramsay-Hunt syndrome [20]. Given the known neuropathogenicity of HSV-1 and the presence of HSV-1 DNA in the lesional site of the facial nerve specific to patients with Bell palsy, we conclude that HSV-1 infection in the facial nerve is directly related to the pathogenesis of Bell palsy just as the varicella-zoster virus is directly related to the pathogenesis of the Ramsay-Hunt syndrome. There are two possible explanations for our failure to detect HSV-1 in three of the patients with Bell palsy [patients 9, 12, and 13]: 1) the limited sensitivity of PCR analysis to detect small amounts of viral DNA and 2) the presence of an etiologic agent other than HSV-1. More data are required to determine the percentage of patients with Bell palsy in whom HSV-1 is the etiologic agent of Bell palsy, but our findings suggest that HSV-1 infection is the major cause of Bell palsy and that treatment with appropriate antiviral agents might benefit most patients with this condition. Drs. Mizobuchi, Nakashiro, and Doi: Department of Neuropsychiatry, Ehime Universit

Tissue‐Specific Expression of Isoaspartyl Protein Carboxyl Methyltransferase Gene in Rat Brain and Testis

Abstract: Isoaspartyl protein carboxyl methyltransferase (PIMT) is widely distributed in mammalian tissues. Using a polymerase chain reaction‐generated 124‐bp DNA fragment from brain cDNA as a probe, four different sizes (∼4.0, 2.5, 1.7, and 1.1 kb) of transcripts were detected with northern blot analysis. They were expressed predominantly in rat brain and testis. The major transcripts were 2.5 and 1.7 kb in the brain and 2.5 and 1.1 kb in the testis. One of the major transcripts specific to the testis (1.1 kb) was determined to study the structural difference of major transcripts in the two tissues. This testicular cDNA had neither the 5’(94 nucleotides) nor the 3’(594 nucleotides) end of previously reported brain cDNA corresponding to 1.7 kb. The mRNA levels and enzyme activities of different regions and developmental changes were examined in the brain. The mRNA levels and enzyme activities were concomitantly high in cerebral cortex and hippocampus. Although they increased rapidly ~30 days after birth in the testis and decreased in aged rats, they increased gradually after birth and remained high during the aging of the brain. Both structural and developmental studies show that the expression of the PIMT gene in brain and testis is regulated in a tissue‐specific manner.

Varicella-Zoster Virus Distribution in Ramsay Hunt Syndrome Revealed by Polymerase Chain Reaction

The pathogenesis of facial nerve paralysis and vestibulo-cochlear dysfunction of Ramsay Hunt syndrome remains unclear as varicella-zoster virus (VZV) has not been demonstrated in the lesions. Using the polymerase chain reaction, we detected VZV genomes not only in the vesicles on the auricles or oral cavity but also in the facial nerve sheath, middle ear mucosa and cerebrospinal fluid from patients with Ramsay Hunt syndrome. The VZV genome was undetectable in the same kinds of clinical samples obtained from control patients with facial nerve paralysis of other etiologies. The results indicated that VZV spreads widely in the neural components, mucocutaneous tissue and cerebrospinal fluid. The present study will facilitate better understanding of the pathogenesis of facial nerve paralysis, vertigo, hearing impairment and other cranial nerve dysfunction of Ramsay Hunt syndrome.

Disappearance of circadian rhythms in Parkinson's disease model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in dogs

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to dogs produces clinical, pathological and neurological features in dog resembling human Parkinsons disease. Using this animal model, we studied the changes in diurnal rhythms of urine volume, creatinine in urine, and vasopressin, aldosterone and renin activity in plasma. Before MPTP treatment, urine volume showed a peak between 17.00 and 1.00 and plasma vasopressin concentration also showed a clear circadian rhythm with a peak at 13.00 and a minimum level at 5.00. Two weeks after MPTP treatment (2.5 mg/kg i.v.), the rhythm of urine volume disappeared and that of vasopressin became less clear. Plasma renin activity increased 2 and 4 weeks after MPTP treatment. The increase was, however, not enough to change the concentration of plasma aldosterone. We examined the effect of L-3,4-dihydroxyphenylalanine (levodopa), on the circadian pattern of urine volume and vasopressin attenuated by MPTP. Levodopa (4 mg/kg/day) was administered orally every day from the first week after MPTP treatment. The circadian rhythms of urine volume and vasopressin reappeared within one week after the start of levodopa administration.

Role of Herpes Simplex Virus Infection in the Pathogenesis of Facial Paralysis in Mice

To clarify the role and site of herpes simplex virus (HSV) infection in the pathogenesis of facial paralysis, we examined the viral genome by the polymerase chain reaction and the neutralization antibody titer using microplates in an animal model. Following inoculation with HSV type 1 of the KOS strain into mouse auricles, HSV DNA appeared in the ipsilateral facial nerve on the 3rd day, and in bilateral facial nerves and the brain stem on the 10th day only in animals with facial paralysis. In animals without facial paralysis, no HSV DNA was detected in these tissues. The neutralization antibody titer was elevated between 4 and 20 days in all animals, with or without facial paralysis. Facial paralysis developed only on the inoculated side, even though HSV DNA was also present in the contralateral facial nerve. We conclude that HSV infection in the facial nerve and brain stem is prerequisite for facial paralysis, and suggest that an immunologic reaction following viral infection plays a key role in the pathogenesis.

Increase of plasma adrenocorticotrophin and cortisol in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated dogs

Neuroendocrine abnormality of the hypothalamic-pituitary-adrenal axis was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated dogs as a model of Parkinsons disease. Blood samples were collected every 4 h for 3-4 days before MPTP treatment, around 2 and 4 weeks after the treatment of MPTP (2.5 mg/kg). Adrenocorticotrophin (ACTH) and cortisol concentrations in plasma were determined by radioimmunoassay. The concentrations of plasma ACTH and cortisol increased by 40% and 60% after MPTP treatment, respectively. Circadian rhythms of neither plasma ACTH nor cortisol concentrations were observed in both MPTP-treated and control dogs. This high hypothalamic-pituitary-adrenal function in MPTP-treated dogs suggests that MPTP may influence the activity of the hypothalamic neurons of the dog.

Hampered expression of isoaspartyl protein carboxyl methyltransferase gene in the human cataractous lens

Isoaspartyl protein carboxyl methyltransferase (PIMT) is implicated in the repair of age-damaged proteins by converting altered aspartic acid residues to normal L-aspartic acid residues. Northern blot and reverse transcription (RT)-PCR analyses have revealed that PIMT gene expression in the human lens is detected exclusively in epithelial cells, and that the mRNA levels in cataractous lens epithelia are significantly lower than those in normal age-matched lens tissue. These results suggest that PIMT may play a vital role in maintaining the clarity of the lens and preventing cataract formation.

Accelerated protein turnover in the skeletal muscle of dystrophic mice

The excretion of 3-methylhistidine increased in the urine of dystrophic mice C57BL/6J. The content of 3-methylhistidine residue decreased in the muscle proteins of dystrophic mice, but not in other organs. Methylated proteins in the skeletal muscle, actin and myosin, were partially purified from the dystrophic and control muscles. The amount of 3-methylhistidine residue in unit weight of the actin and myosin preparations was normal in dystrophic muscle. These three facts indicate that the turnover rates of actin and myosin are increased in the muscle of the dystrophic mice.

Role of T-lymphocyte subsets in facial nerve paralysis owing to the reactivation of herpes simplex virus type 1

Conclusion. Although both T-cell subsets are essential for inhibiting HSV-1 reactivation in the GG, CD4+ T cells play a more important role in host defense against virus replication. Objective. To elucidate the host immunological factors that participate in herpes simplex virus type 1 (HSV-1) reactivation in the geniculate ganglia (GG) and lead to facial paralysis, we developed a mouse model of facial paralysis that involved the reactivation of HSV-1 following general immune suppression. Material and methods. Eight weeks after recovery from primary facial paralysis caused by inoculating the auricle with HSV-1 the auricle was scratched and mice (n=69) were given an i.p. injection of either anti-CD4 (n=46) or anti-CD8 (n=23) monoclonal antibody to deplete specific T-lymphocyte subsets. Following this reactivation procedure, the rate of recurrent facial paralysis was compared between the two models. The GG were examined histopathologically and using polymerase chain reaction to detect HSV-1 DNA. Results. Facial paralysis developed in 42% of mice in the anti-CD4 model and in 13% in the anti-CD8 model. HSV-1 DNA was detected in 50% of the mice in both models. Histopathologically, neurons were destroyed in parts of the GG and numerous virus particles were seen in the surviving neurons.

Day-Night Variation of Urine Volume in Parkinson's Disease

Abstract: Studies on the circadian rhythm of urine excretion in healthy men have demonstrated that the maximal urine flow occurs in the early afternoon and the minimal around midnight. In this study, an abnormality in the variation of urine volume was found in parkinsonian patients. Urine samples were collected during daytime (9:00–21:00) and nighttime (21:00–9:00). Fifteen healthy control subjects were examined and found to excrete 60% during the daytime and 40% during the nighttime of the total urine volume. Sixteen parkinsonian patients excreted 43% during the daytime and 57% during the nighttime. In contrast to the control subjects, the parkinsonian patients excreted a smaller volume of their urine during the daytime than during the nighttime. This finding might be related to the degeneration of dopaminergic and/or nondopaminergic neurons in the brain which control urinary excretion.