The Laryngoscope C 2014 The American Laryngological, V
Rhinological and Otological Society, Inc.
Facial Nerve Palsy After Reactivation of Herpes Simplex Virus Type 1 in Diabetic Mice Shinichi Esaki, MD, PhD; Koji Yamano, MD, PhD; Sachiyo Katsumi, MD; Toshiya Minakata, MD; Shingo Murakami, MD, PhD Objectives/Hypothesis: Bell’s palsy is highly associated with diabetes mellitus (DM). Either the reactivation of herpes simplex virus type 1 (HSV-1) or diabetic mononeuropathy has been proposed to cause the facial paralysis observed in DM patients. However, distinguishing whether the facial palsy is caused by herpetic neuritis or diabetic mononeuropathy is difficult. We previously reported that facial paralysis was aggravated in DM mice after HSV-1 inoculation of the murine auricle. In the current study, we induced HSV-1 reactivation by an auricular scratch following DM induction with streptozotocin (STZ). Study Design: Controlled animal study. Methods: Diabetes mellitus was induced with streptozotocin injection in only mice that developed transient facial nerve paralysis with HSV-1. Recurrent facial palsy was induced after HSV-1 reactivation by auricular scratch. Results: After DM induction, the number of cluster of differentiation 3 (CD3)1 T cells decreased by 70% in the DM mice, and facial nerve palsy recurred in 13% of the DM mice. Herpes simplex virus type 1 deoxyribonucleic acid (DNA) was detected in the facial nerve of all of the DM mice with palsy, and HSV-1 capsids were found in the geniculate ganglion using electron microscopy. Herpes simplex virus type 1 DNA was also found in some of the DM mice without palsy, which suggested the subclinical reactivation of HSV-1. Conclusions: These results suggested that HSV-1 reactivation in the geniculate ganglion may be the main causative factor of the increased incidence of facial paralysis in DM patients. Key Words: Bell’s palsy, herpes simplex virus, diabetes mellitus, HSV-1 reactivation. Level of Evidence: N/A. Laryngoscope, 125:E143–E148, 2015
INTRODUCTION Bell’s palsy is defined as idiopathic, acute unilateral peripheral facial palsy.1 It accounts for approximately 60% to 75% of all acute peripheral facial palsy. Recent molecular biological studies strongly suggested that the reactivation of herpes simplex virus type 1 (HSV-1) was the main cause of Bell’s palsy.2–5 Epidemiological studies have also demonstrated that Bell’s palsy is highly associated with diabetes mellitus (DM).6–8 Cranial neuropathy is rare, and ocular neuropathy is the most common cranial neuropathy. Patients with DM can develop diplopia from isolated oculomotor (third), trochlear (fourth), or abducens (sixth) nerve palsies—or from combined ocular nerve palsies. Regardless of the severity, the vast majority of patients with diabetic ocular motor palsy recover completely within 3 to 6 From the Department of Otolaryngology, Head and Neck Surgery, Nagoya City University Graduate School of Medical Sciences and Medical School (S.E., K.Y., S.K., T.M., S.M.); and from the Department of Virology, Graduate School of Medicine, Nagoya University (S.E.), Nagoya, Japan. Editor’s Note: This Manuscript was accepted for publication September 30, 2014. This study was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan (23791919, 25861579). The authors have no other funding, financial relationships, or conflicts of interest to disclose. Send correspondence to S. Esaki, MD, Department of Otolaryngology, Head and Neck Surgery, Nagoya City University Graduate School of Medical Sciences and Medical School, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8602, Japan. E-mail: [email protected] DOI: 10.1002/lary.24994
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months.9 Microvascular ischemia from DM is the most common etiology for ocular neuropathy, and the microvascular ischemia can cause peripheral nerve neuropathy similar to Bell’s palsy. Thus, it is unknown whether the facial palsy observed in DM patients is caused by diabetic mononeuropathy or by the reactivation of HSV-1, and it is unclear whether DM patients with peripheral facial nerve palsy should be regarded as having Bell’s palsy or diabetic cranial neuropathy. Previously, we reported that facial paralysis developed more frequently in DM mice that were inoculated with HSV-1 in the auricle.10 The suppression of T-cell immunity plays an important role in the reactivation of herpes viruses,11,12 and T-cell immunity is significantly suppressed in both DM patients and Bell’s palsy patients.13 These two observations led to the hypothesis that HSV-1 reactivation frequently occurs in DM patients and causes facial nerve paralysis. To address the question of whether the facial palsy in DM patients is caused by diabetic mononeuropathy itself or by the reactivation of HSV-1, we investigated HSV-1 reactivation in a DM mouse model and show that this reactivation resulted in facial nerve palsy.
MATERIALS AND METHODS Animals and Viruses Four-week-old specific–pathogen-free female Balb/c mice (CLEA Inc., Tokyo, Japan) were used throughout this study. All mice were given food and water ad libitum and were cared for
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Flow Cytometric Analysis of T Cells in the Spleen
Fig. 1. Experimental protocol and course of facial nerve paralysis after virus inoculation. HSV-1 was inoculated when the mice were 4 weeks old. Streptozotocin or vehicle was intraperitoneally injected when the mice were 7 weeks old. Auricular scratching was performed on the right ear in mice with and without DM at 8 weeks after DM induction, and the mice were observed thereafter. DM 5 diabetes mellitus; HSV-1 5 herpes simplex virus type 1. in compliance with the guidelines for animal experimentation at Nagoya City University (H21-M56). The KOS strain of HSV1 (1.0 3 105 plaque forming units/25 mL) was inoculated into the right auricle, as described previously.14 Briefly, under ketamine hydrochloride anesthesia (50 mg/kg), the posterior surface of the auricle was scratched 20 times with a 27-gauge needle, and the scratched area was inoculated with HSV-1.
Induction of Recurrent Facial Nerve Paralysis in DM Mice More than half of the mice developed facial nerve paralysis and recovered within 7 days. Only mice that developed transient facial nerve paralysis after the primary infection were used for the following experiments. After a week of recovery from the primary paralysis (at the age of 7 weeks), DM was induced with a single intraperitoneal injection of streptozotocin (STZ) (Sigma, St. Louis, MO) at a dose of 100 mg/kg, which was dissolved in a 0.05-M citric acid solution with a pH of 4.5. Diabetes mellitus was confirmed by blood glucose levels above 250 mg/dL in consecutive measurements.10 For non-DM mice, the same volume of sterile citric acid solution alone was injected. Eight weeks after DM induction (at the age of 15 weeks), HSV-1 reactivation was induced using a 27-gauge needle to scratch the surface of the right auricle where the HSV-1 had been previously inoculated. Thereafter, the mice were observed daily for the appearance of facial nerve paralysis and changes in their general condition. As controls, we also prepared DM mice without scratches and non-DM mice with scratches. The protocol for the experiment is summarized in Figure 1.
Evaluation of Facial Nerve Paralysis After HSV-1 inoculation or the induction of reactivation, the general condition, blink reflex, and vibrissae movement of the mice were monitored daily. Facial nerve paralysis was evaluated following previously described criteria.10 The blink reflex was evoked twice by blowing air onto each eye using an 18gauge needle with a 5-ml syringe and was scored on a scale of 0 to 2 based on the inoculated side. Zero indicated no difference compared with the normal side; 1 indicated a delayed reflex; and 2 indicated a complete loss of reflex. Vibrissae movement was observed for 30 seconds and scored on a scale of 0 to 2. Zero indicated no difference compared with the normal side; 1 indicated weaker movement; and 2 indicated a complete inability to move. Facial nerve function was determined by adding the scores for the blink reflex and vibrissae movement. Therefore, a total score of 3 or 4 indicated severe paralysis; 2 indicated moderate paralysis; 1 indicated light paralysis; and 0 indicated normal function.
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Eight weeks after DM induction (at the age of 15 weeks), eight DM and eight non-DM mice were deeply anesthetized. Splenocytes were isolated, and single cells were subjected to flow cytometry.15 Lymphocytes were collected using Lymphosepar II (Immuno-Biological Laboratories Co., Ltd., Fujioka, Japan). The splenocytes were then washed twice with 1 mL of phosphate-buffered saline (PBS) containing 0.5% fetal calf serum (FCS) and incubated with 0.25 mg of the following monoclonal antibodies diluted in PBS for 20 minutes in the dark: fluorescein isothiocyanate-conjugated antimouse cluster of differentiation 3 (CD3) (clone 17A2) and phycoerythrinconjugated antimouse CD45R/B220 (clone RA3–6B2) (BioLegend, San Diego, CA). After two washes with PBS, the splenocytes were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
PCR of HSV-1 DNA from the Intratemporal Portion of the Facial Nerve Ten days after the induction of reactivation, DM mice with auricular scratching, DM mice without scratching, and non-DM mice with scratching were deeply anesthetized by the intraperitoneal administration of ketamine (150 mg/kg) and xylazine (15 mg/kg) and were euthanized by transcardial perfusion with 50 mL of PBS. The intratemporal portions of both facial nerves, including the geniculate ganglia, were then dissected under a microscope. Deoxyribonucleic acid (DNA) was extracted using a DNeasy tissue kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Synthetic primers encoding part of the virion glycoprotein C (UL44: 50 -CCACCGAGCGGCAGGTGATC-30 ) and the hydrophobic N terminus located on the HSV-1 gene (UL45: 50 -GCCGACCGCCTGCTCGTGCT-30 ) were used for polymerase chain reaction (PCR) amplification.16 The DNA was amplified in a thermal cycler using the following program for 35 cycles: denaturation at 95 C for 90 seconds, annealing at 60 C for 90 seconds, and extension at 73 C for 5 minutes. The PCR products were subjected to electrophoresis on a 1.5% agarose gel. The DNA purified from HSV-1-infected Vero cells was used as a positive control, and distilled water was used as a negative control.
Transmission Electron Microscopy Analysis Ten days after the induction of reactivation, DM mice with auricular scratching, DM mice without scratching, and non-DM mice with scratching were deeply anesthetized and perfused transcardially with 2.5% formaldehyde and 2.5% glutaraldehyde. The intratemporal portion of the facial nerve was
TABLE I. Recurrence of Facial Nerve Palsy. Mice
Auricular Scratch
Paralysis
Death
DM
1
4/30* (13.3%)
5/30* (16.7%)
DM
2
0/30
0/30
1
(0%) 0/30
(0%) 0/30
(0%)
(0%)
Non-DM
*P < 0.001 compared with the other groups. DM 5 diabetes mellitus.
Esaki et al.: Facial Nerve Palsy After Reactivation of HSV-1 in DM Mice
Fig. 2. (A) Representative picture of the flow cytometric analysis of CD31/ B2202 T cells from mice with or without DM at 8 weeks after streptozotocin injection. (B) Flow cytometric analysis of CD31/ B220- T cells from the splenocytes of mice with or without DM (n 5 8). Compared with the non-DM mice, CD31/ B2202 T cells were significantly decreased (*P < 0.01). CD3 5 cluster of differentiation 3; DM 5 diabetes mellitus.
resected, postfixed with 2% osmium tetroxide, dehydrated, and ˚embedded in epoxy resin. The specimens were sliced into 700-A thick sections, stained with uranyl acetate-lead citrate, and evaluated using transmission electron microscopy (JEM1200EX, JEOL Ltd., Tokyo, Japan).
Statistical Analyses The facial nerve score data were evaluated using the Mann-Whitney test, and the remaining data were evaluated with unpaired t tests using StatView Software (SAS Institute, Cary, NC). The number of T cells was analyzed using the unpaired t test, and the remaining data were analyzed using one-way factorial analysis of variance followed by the Tukey test for multiple comparisons among groups. Differences with a probability value of P < 0.05 were considered statistically significant.
RESULTS Recurrent Facial Nerve Paralysis after Auricular Scratch in DM Mice Herpes simplex virus type 1 was inoculated on the right side in 210 mice, and 109 mice (51.9%) developed facial nerve paralysis on the inoculated side 6 to 8 days postinfection. The paralysis persisted for a week and recovered spontaneously in all cases. In a previous study, latent HSV-1 was detected from the geniculate ganglia of the mice that recovered from facial palsy.16 Ninety mice were selected randomly from the 109 mice and divided into three groups before the injection of STZ or citric acid. The induction of reactivation was performed using auricular scratching in 30 DM mice at 8 weeks after DM induction. Among these 30 mice, four had moderate facial nerve palsy on the right side 5 to 7 days after the reactivation induction (Table I). Five mice showed inactivity 2 to 5 days after the reactivation induction, and all of these mice died from encephalitis. Meanwhile, neither facial nerve paralysis nor death was observed in the DM mice without auricular scratching (n 5 30) or in the non-DM mice with auricular scratching (n 5 30). Laryngoscope 125: April 2015
Decrease of CD31/B2202 T Cells in the Spleen of DM Mice Cell-mediated immunity plays a major role in host defense against HSV-1.11 Therefore, we analyzed CD31 T cells in the spleens of mice with and without DM. Lymphocytes from the spleen were collected at 8 weeks after DM induction. The CD31/B2202 T-cell levels were significantly decreased in the DM mice compared with the non-DM mice (P < 0.01; Fig. 2A and 2B).
PCR Evaluation of Reactivated HSV-1 in the Facial Nerve The intratemporal portion of the facial nerve was resected and examined for HSV-1 DNA by PCR. Herpes simplex virus type 1 DNA was found in six of 15 DM mice after the reactivation induction, and two of these mice had facial nerve palsy on the right side (Table II). As shown in Figure 3, HSV-1 DNA was detected in two mice (4 and 5) with paralysis and in one mouse (1) without paralysis on the right side. Meanwhile, no HSV-1 DNA was detected in DM mice without auricular scratching or in non-DM mice with auricular scratching.
TABLE II. HSV-1 DNA from the Intratemporal Portion of the Facial Nerve. Mice
Auricular Scratch
Right (inoculated)
Left
DM
1
6/15 * (40.0%)
0/15 (0%)
DM
2
0/15
0/15
1
(0%) 0/15
(0%) 0/15
(0%)
(0%)
Non-DM
*P < 0.001 compared with the other groups. DM 5 diabetes mellitus.
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Fig. 3. Polymerase chain reaction analyses of the HSV-1 genomes from the bilateral facial nerves of DM mice at 10 days after auricular scratching (n 5 5). Three mice (1, 2, and 3) had no facial palsy, whereas two mice (4 and 5) had facial nerve palsy on the right side. The HSV-1 genome was detected in three mice (1, 4, and 5) on the right side. DM 5 diabetes mellitus; L 5 left facial nerve; N 5 negative control; P 5 positive control; R 5 right facial nerve; HSV-1 5 herpes simplex virus type 1.
Fig. 4. Electron microscopy images of the right intratemporal facial nerve and right geniculate ganglion at 10 days after auricular scratching. (a) Facial nerve of a DM mouse with paralysis after auricular scratching. The facial nerve was edematous, and myelin degeneration was observed. Some fibers were demyelinated with vascular changes in Schwann cells. (b) Facial nerve of a DM mouse without paralysis after auricular scratching, showing slight myelin degeneration. (c) Facial nerve of a non-DM mouse after auricular scratching, showing no myelin degeneration. (d) Geniculate ganglion of a DM mouse with palsy. Numerous HSV-1 capsids were observed in the rough endoplasmic reticulum. (e) Magnified image of the geniculate ganglion, showing HSV-1 capsids (arrows). Scale bars indicate 2 mm (a, b, and c), 500 nm (d), and 200 nm (e). DM 5 diabetes mellitus; GG 5 geniculate ganglion; HSV-1 5 herpes simplex virus type 1.
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Esaki et al.: Facial Nerve Palsy After Reactivation of HSV-1 in DM Mice
Electron Microscopic Examination of the Right Facial Nerve and Geniculate Ganglia Finally, we used transmission electron microscopy to examine the right facial nerve and geniculate ganglia of DM mice at 10 days after the induction of HSV-1 reactivation. The facial nerve of a DM mouse with paralysis was edematous and showed myelin degeneration. Some fibers were demyelinated with vascular changes in the Schwann cells (Fig. 4a). No fibers were demyelinated in the facial nerve of a DM mouse without paralysis, but slight myelin degeneration was observed (Fig. 4b). No myelin degeneration was observed in the facial nerve of a DM mouse without scratching (data not shown) or in that of a non-DM mouse with auricular scratching (Fig. 4c). The geniculate ganglia were examined, and numerous HSV-1 particles were observed in some ganglia, which had well-preserved fine structures, of a DM mouse with paralysis (Fig. 4d). Figure 4e shows a magnified image of the geniculate ganglia in which numerous HSV-1 particles can be observed in the rough endoplasmic reticulum.
DISCUSSION The incidence of DM is 10.5% in Japan, as reported by the Ministry of Health, Labor, and Welfare of Japan, whereas the incidence of DM in Bell’s palsy patients is more than 20%.17 This suggests that facial nerve paralysis develops more frequently in DM patients. Bell’s palsy is mainly caused by latent HSV-1 in the geniculate ganglion of the facial nerve.1,2 However, DM is closely related to cranial mononeuropathy and segmental motor paralysis.18–20 Thus, if DM patients develop facial nerve palsy, whether HSV-1 or DM is the cause of the facial palsy is unclear. This issue is important when we use steroids for medical treatment because steroid administration can exacerbate the conditions of DM patients. Neuropathy of the third cranial nerve is thought to be caused by diabetic mononeuropathy or angiopathy because it develops frequently in severe DM.21 Bell’s palsy is also more frequently observed in DM patients; however, it can develop regardless of the severity of DM, and the prognosis of facial nerve palsy does not differ between patients with and without DM.6 These differences suggest that the causes of the neuropathy of the third and seventh nerves could be different. Previously, we reported that facial paralysis induced by HSV-1 occurred more frequently in DM mice.10 Considering that DM amplifies the infectivity of HSV-1, we hypothesized that Bell’s palsy could easily be caused by the reactivation of HSV-1 under DM. In the present study, we investigated HSV-1 reactivation in DM mice and succeeded in inducing facial nerve paralysis. Mild skin trauma to the ear could induce HSV-1 reactivation in the murine trigeminal nerve.22 In contrast, skin trauma alone was not enough to induce HSV1 reactivation in the murine facial nerve. A previous study demonstrated that facial nerve paralysis could be induced after auricular scratching followed by the depletion of CD31 cells via an intraperitoneal injection of anti-CD3 monoclonal antibody.16 In DM patients, CD31, Laryngoscope 125: April 2015
CD41, CD81/CD282, and CD81/CD281 lymphocytes are significantly decreased compared with their levels in non-DM patients.13 Peripheral T lymphocytes were significantly decreased in patients with Bell’s palsy in the early stage of the disease.23,24 Both helper and cytotoxic T cells play major roles in host defense against HSV-111 and are thought to inhibit the spread of reactivated HSV-1. In the present study, recurrent facial nerve palsy could develop in DM mice after auricular scratching alone. After DM induction, CD31 cells decreased by 70%, which could accelerate development of facial nerve palsy after auricular scratching. The HSV-1 DNA was found in some of the DM mice without facial nerve paralysis and in the DM mice with paralysis. Electron microscopy also showed reactivated HSV-1 capsids in the geniculate ganglia of the DM mice with paralysis. Auricular scratching did induce HSV-1 reactivation in some DM mice, and facial nerve paralysis was observed in a subset of these DM mice with reactivated HSV-1 in the geniculate ganglia. A previous study also reported that the incidence of HSV-1 reactivation (67%) was more frequent than that of facial nerve paralysis (20%).16 Interestingly, the ratio of paralysis to HSV1 reactivation in the previous study (20%:67%) was similar to that in the current study (13%:40%). In the previous study, evoked electromyography remained at 60% to 70% when facial nerve palsy had recovered completely.10 We assume that the subclinical reactivation of HSV-1 disturbed the facial nerve to some extent but failed to develop into paralysis.
CONCLUSION We induced recurrent facial nerve paralysis in DM mice. The recurrent paralysis occurred in the DM mice only after auricular scratching. Auricular scratching and a decrease in CD31 T cells in the presence of DM induced the reactivation of HSV-1 in the geniculate ganglia, which resulted in facial nerve paralysis. Here, we propose that facial nerve paralysis in patients with DM arises as the result of HSV-1 reactivation, as is the case in Bell’s palsy, rather than as the result of a diabetic disorder.
BIBLIOGRAPHY 1. Gilden DH. Clinical practice. Bell’s Palsy. N Engl J Med 2004;351:1323– 1331. 2. Murakami S, Mizobuchi M, Nakashiro Y, Doi T, Hato N, Yanagihara N. Bell palsy and herpes simplex virus: identification of viral DNA in endoneurial fluid and muscle. Ann Intern Med 1996;124:27–30. 3. Abiko Y, Ikeda M, Hondo R. Secretion and dynamics of herpes simplex virus in tears and saliva of patients with Bell’s palsy. Otol Neurotol 2002;23:779–783. 4. Furuta Y, Fukuda S, Chida E, et al. Reactivation of herpes simplex virus type 1 in patients with Bell’s palsy. J Med Virol 1998;54:162–166. 5. Turriziani O, Falasca F, Maida P, et al. Early collection of saliva specimens from Bell’s palsy patients: Quantitative analysis of HHV-6, HSV-1, and VZV. J Med Virol 2014;86:1752–1758. 6. Yanagihara N, Hyodo M. Association of diabetes mellitus and hypertension with Bell’s palsy and Ramsay Hunt syndrome. Ann Otol Rhinol Laryngol Suppl 1988;137:5–7. 7. Adour KK, Bell DN, Wingerd J. Bell palsy. Dilemma of diabetes mellitus. Arch Otolaryngol 1974;99:114–117. 8. Bosco D, Plastino M, Bosco F, et al. Bell’s palsy: a manifestation of prediabetes? Acta Neurol Scand 2011;123:68–72. 9. Greco D, Gambina F, Maggio F. Ophthalmoplegia in diabetes mellitus: a retrospective study. Acta Diabetol 2009;46:23–26.
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10. Esaki S, Yamano K, Kiguchi J, et al. Diabetic mice show an aggravated course of herpes-simplex virus-induced facial nerve paralysis. Otol Neurotol 2012;33:1452–1457. 11. Schmid DS, Rouse BT. Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors. J Immunol 1983;131:479–484. 12. Mojadadi S, Jamali A, Khansarinejad B, Soleimanjahi H, Bamdad T. Acute morphine administration reduces cell-mediated immunity and induces reactivation of latent herpes simplex virus type 1 in BALB/c mice. Cell Mol Immunol 2009;6:111–116. 13. Dworacka M, Winiarska H, Borowska M, Abramczyk M, BobkiewiczKozlowska T, Dworacki G. Pro-atherogenic alterations in T-lymphocyte subpopulations related to acute hyperglycaemia in type 2 diabetic patients. Circ J 2007;71:962–967. 14. Sugita T, Murakami S, Yanagihara N, Fujiwara Y, Hirata Y, Kurata T. Facial nerve paralysis induced by herpes simplex virus in mice: an animal model of acute and transient facial paralysis. Ann Otol Rhinol Laryngol 1995;104:574–581. 15. Esaki S, Goshima F, Kimura H, Murakami S, Nishiyama Y. Enhanced antitumoral activity of oncolytic herpes simplex virus with gemcitabine using colorectal tumor models. Int J Cancer 2013;132:1592–1601.
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16. Takahashi H, Hitsumoto Y, Honda N et al. Mouse model of Bell’s palsy induced by reactivation of herpes simplex virus type 1. J Neuropathol Exp Neurol 2001;60:621–627. 17. Kanazawa A, Haginomori S, Takamaki A, Nonaka R, Araki M, Takenaka H. Prognosis for Bell’s palsy: a comparison of diabetic and nondiabetic patients. Acta Otolaryngol 2007;127:888–891. 18. Watanabe K, Hagura R, Akanuma Y, et al. Characteristics of cranial nerve palsies in diabetic patients. Diabetes Res Clin Pract 1990;10:19–27. 19. Raff MC, Asbury AK. Ischemic mononeuropathy and mononeuropathy multiplex in diabetes mellitus. N Engl J Med 1968;279:17–21. 20. Acaroglu G, Akinci A, Zilelioglu O. Retinopathy in patients with diabetic ophthalmoplegia. Ophthalmologica 2008;222:225–228. 21. Hopf HC, Gutmann L. Diabetic 3rd nerve palsy: evidence for a mesencephalic lesion. Neurology 1990;40:1041–1045. 22. Hill TJ, Blyth WA, Harbour DA. Trauma to the skin causes recurrence of herpes simplex in the mouse. J Gen Virol 1978;39:21–28. 23. Aviel A, Ostfeld E, Burstein R, Marshak G, Bentwich Z. Peripheral blood T and B lymphocyte subpopulations in Bell’s palsy. Ann Otol Rhinol Laryngol 1983;92:187–191. 24. Tekgul H, Polat M, Serdaroglu G, et al. Lymphocyte subsets in Bell’s palsy: immune pathogenesis and outcome prediction. Pediatr Neurol 2004;31:258–260.
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Facial Nerve Palsy After Reactivation of Herpes Simplex Virus Type 1 in Diabetic Mice Shinichi Esaki, MD, PhD; Koji Yamano, MD, PhD; Sachiyo Katsumi, MD; Toshiya Minakata, MD; Shingo Murakami, MD, PhD Objectives/Hypothesis: Bell’s palsy is highly associated with diabetes mellitus (DM). Either the reactivation of herpes simplex virus type 1 (HSV-1) or diabetic mononeuropathy has been proposed to cause the facial paralysis observed in DM patients. However, distinguishing whether the facial palsy is caused by herpetic neuritis or diabetic mononeuropathy is difficult. We previously reported that facial paralysis was aggravated in DM mice after HSV-1 inoculation of the murine auricle. In the current study, we induced HSV-1 reactivation by an auricular scratch following DM induction with streptozotocin (STZ). Study Design: Controlled animal study. Methods: Diabetes mellitus was induced with streptozotocin injection in only mice that developed transient facial nerve paralysis with HSV-1. Recurrent facial palsy was induced after HSV-1 reactivation by auricular scratch. Results: After DM induction, the number of cluster of differentiation 3 (CD3)1 T cells decreased by 70% in the DM mice, and facial nerve palsy recurred in 13% of the DM mice. Herpes simplex virus type 1 deoxyribonucleic acid (DNA) was detected in the facial nerve of all of the DM mice with palsy, and HSV-1 capsids were found in the geniculate ganglion using electron microscopy. Herpes simplex virus type 1 DNA was also found in some of the DM mice without palsy, which suggested the subclinical reactivation of HSV-1. Conclusions: These results suggested that HSV-1 reactivation in the geniculate ganglion may be the main causative factor of the increased incidence of facial paralysis in DM patients. Key Words: Bell’s palsy, herpes simplex virus, diabetes mellitus, HSV-1 reactivation. Level of Evidence: N/A. Laryngoscope, 125:E143–E148, 2015
INTRODUCTION Bell’s palsy is defined as idiopathic, acute unilateral peripheral facial palsy.1 It accounts for approximately 60% to 75% of all acute peripheral facial palsy. Recent molecular biological studies strongly suggested that the reactivation of herpes simplex virus type 1 (HSV-1) was the main cause of Bell’s palsy.2–5 Epidemiological studies have also demonstrated that Bell’s palsy is highly associated with diabetes mellitus (DM).6–8 Cranial neuropathy is rare, and ocular neuropathy is the most common cranial neuropathy. Patients with DM can develop diplopia from isolated oculomotor (third), trochlear (fourth), or abducens (sixth) nerve palsies—or from combined ocular nerve palsies. Regardless of the severity, the vast majority of patients with diabetic ocular motor palsy recover completely within 3 to 6 From the Department of Otolaryngology, Head and Neck Surgery, Nagoya City University Graduate School of Medical Sciences and Medical School (S.E., K.Y., S.K., T.M., S.M.); and from the Department of Virology, Graduate School of Medicine, Nagoya University (S.E.), Nagoya, Japan. Editor’s Note: This Manuscript was accepted for publication September 30, 2014. This study was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan (23791919, 25861579). The authors have no other funding, financial relationships, or conflicts of interest to disclose. Send correspondence to S. Esaki, MD, Department of Otolaryngology, Head and Neck Surgery, Nagoya City University Graduate School of Medical Sciences and Medical School, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8602, Japan. E-mail: [email protected] DOI: 10.1002/lary.24994
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months.9 Microvascular ischemia from DM is the most common etiology for ocular neuropathy, and the microvascular ischemia can cause peripheral nerve neuropathy similar to Bell’s palsy. Thus, it is unknown whether the facial palsy observed in DM patients is caused by diabetic mononeuropathy or by the reactivation of HSV-1, and it is unclear whether DM patients with peripheral facial nerve palsy should be regarded as having Bell’s palsy or diabetic cranial neuropathy. Previously, we reported that facial paralysis developed more frequently in DM mice that were inoculated with HSV-1 in the auricle.10 The suppression of T-cell immunity plays an important role in the reactivation of herpes viruses,11,12 and T-cell immunity is significantly suppressed in both DM patients and Bell’s palsy patients.13 These two observations led to the hypothesis that HSV-1 reactivation frequently occurs in DM patients and causes facial nerve paralysis. To address the question of whether the facial palsy in DM patients is caused by diabetic mononeuropathy itself or by the reactivation of HSV-1, we investigated HSV-1 reactivation in a DM mouse model and show that this reactivation resulted in facial nerve palsy.
MATERIALS AND METHODS Animals and Viruses Four-week-old specific–pathogen-free female Balb/c mice (CLEA Inc., Tokyo, Japan) were used throughout this study. All mice were given food and water ad libitum and were cared for
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Flow Cytometric Analysis of T Cells in the Spleen
Fig. 1. Experimental protocol and course of facial nerve paralysis after virus inoculation. HSV-1 was inoculated when the mice were 4 weeks old. Streptozotocin or vehicle was intraperitoneally injected when the mice were 7 weeks old. Auricular scratching was performed on the right ear in mice with and without DM at 8 weeks after DM induction, and the mice were observed thereafter. DM 5 diabetes mellitus; HSV-1 5 herpes simplex virus type 1. in compliance with the guidelines for animal experimentation at Nagoya City University (H21-M56). The KOS strain of HSV1 (1.0 3 105 plaque forming units/25 mL) was inoculated into the right auricle, as described previously.14 Briefly, under ketamine hydrochloride anesthesia (50 mg/kg), the posterior surface of the auricle was scratched 20 times with a 27-gauge needle, and the scratched area was inoculated with HSV-1.
Induction of Recurrent Facial Nerve Paralysis in DM Mice More than half of the mice developed facial nerve paralysis and recovered within 7 days. Only mice that developed transient facial nerve paralysis after the primary infection were used for the following experiments. After a week of recovery from the primary paralysis (at the age of 7 weeks), DM was induced with a single intraperitoneal injection of streptozotocin (STZ) (Sigma, St. Louis, MO) at a dose of 100 mg/kg, which was dissolved in a 0.05-M citric acid solution with a pH of 4.5. Diabetes mellitus was confirmed by blood glucose levels above 250 mg/dL in consecutive measurements.10 For non-DM mice, the same volume of sterile citric acid solution alone was injected. Eight weeks after DM induction (at the age of 15 weeks), HSV-1 reactivation was induced using a 27-gauge needle to scratch the surface of the right auricle where the HSV-1 had been previously inoculated. Thereafter, the mice were observed daily for the appearance of facial nerve paralysis and changes in their general condition. As controls, we also prepared DM mice without scratches and non-DM mice with scratches. The protocol for the experiment is summarized in Figure 1.
Evaluation of Facial Nerve Paralysis After HSV-1 inoculation or the induction of reactivation, the general condition, blink reflex, and vibrissae movement of the mice were monitored daily. Facial nerve paralysis was evaluated following previously described criteria.10 The blink reflex was evoked twice by blowing air onto each eye using an 18gauge needle with a 5-ml syringe and was scored on a scale of 0 to 2 based on the inoculated side. Zero indicated no difference compared with the normal side; 1 indicated a delayed reflex; and 2 indicated a complete loss of reflex. Vibrissae movement was observed for 30 seconds and scored on a scale of 0 to 2. Zero indicated no difference compared with the normal side; 1 indicated weaker movement; and 2 indicated a complete inability to move. Facial nerve function was determined by adding the scores for the blink reflex and vibrissae movement. Therefore, a total score of 3 or 4 indicated severe paralysis; 2 indicated moderate paralysis; 1 indicated light paralysis; and 0 indicated normal function.
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Eight weeks after DM induction (at the age of 15 weeks), eight DM and eight non-DM mice were deeply anesthetized. Splenocytes were isolated, and single cells were subjected to flow cytometry.15 Lymphocytes were collected using Lymphosepar II (Immuno-Biological Laboratories Co., Ltd., Fujioka, Japan). The splenocytes were then washed twice with 1 mL of phosphate-buffered saline (PBS) containing 0.5% fetal calf serum (FCS) and incubated with 0.25 mg of the following monoclonal antibodies diluted in PBS for 20 minutes in the dark: fluorescein isothiocyanate-conjugated antimouse cluster of differentiation 3 (CD3) (clone 17A2) and phycoerythrinconjugated antimouse CD45R/B220 (clone RA3–6B2) (BioLegend, San Diego, CA). After two washes with PBS, the splenocytes were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
PCR of HSV-1 DNA from the Intratemporal Portion of the Facial Nerve Ten days after the induction of reactivation, DM mice with auricular scratching, DM mice without scratching, and non-DM mice with scratching were deeply anesthetized by the intraperitoneal administration of ketamine (150 mg/kg) and xylazine (15 mg/kg) and were euthanized by transcardial perfusion with 50 mL of PBS. The intratemporal portions of both facial nerves, including the geniculate ganglia, were then dissected under a microscope. Deoxyribonucleic acid (DNA) was extracted using a DNeasy tissue kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Synthetic primers encoding part of the virion glycoprotein C (UL44: 50 -CCACCGAGCGGCAGGTGATC-30 ) and the hydrophobic N terminus located on the HSV-1 gene (UL45: 50 -GCCGACCGCCTGCTCGTGCT-30 ) were used for polymerase chain reaction (PCR) amplification.16 The DNA was amplified in a thermal cycler using the following program for 35 cycles: denaturation at 95 C for 90 seconds, annealing at 60 C for 90 seconds, and extension at 73 C for 5 minutes. The PCR products were subjected to electrophoresis on a 1.5% agarose gel. The DNA purified from HSV-1-infected Vero cells was used as a positive control, and distilled water was used as a negative control.
Transmission Electron Microscopy Analysis Ten days after the induction of reactivation, DM mice with auricular scratching, DM mice without scratching, and non-DM mice with scratching were deeply anesthetized and perfused transcardially with 2.5% formaldehyde and 2.5% glutaraldehyde. The intratemporal portion of the facial nerve was
TABLE I. Recurrence of Facial Nerve Palsy. Mice
Auricular Scratch
Paralysis
Death
DM
1
4/30* (13.3%)
5/30* (16.7%)
DM
2
0/30
0/30
1
(0%) 0/30
(0%) 0/30
(0%)
(0%)
Non-DM
*P < 0.001 compared with the other groups. DM 5 diabetes mellitus.
Esaki et al.: Facial Nerve Palsy After Reactivation of HSV-1 in DM Mice
Fig. 2. (A) Representative picture of the flow cytometric analysis of CD31/ B2202 T cells from mice with or without DM at 8 weeks after streptozotocin injection. (B) Flow cytometric analysis of CD31/ B220- T cells from the splenocytes of mice with or without DM (n 5 8). Compared with the non-DM mice, CD31/ B2202 T cells were significantly decreased (*P < 0.01). CD3 5 cluster of differentiation 3; DM 5 diabetes mellitus.
resected, postfixed with 2% osmium tetroxide, dehydrated, and ˚embedded in epoxy resin. The specimens were sliced into 700-A thick sections, stained with uranyl acetate-lead citrate, and evaluated using transmission electron microscopy (JEM1200EX, JEOL Ltd., Tokyo, Japan).
Statistical Analyses The facial nerve score data were evaluated using the Mann-Whitney test, and the remaining data were evaluated with unpaired t tests using StatView Software (SAS Institute, Cary, NC). The number of T cells was analyzed using the unpaired t test, and the remaining data were analyzed using one-way factorial analysis of variance followed by the Tukey test for multiple comparisons among groups. Differences with a probability value of P < 0.05 were considered statistically significant.
RESULTS Recurrent Facial Nerve Paralysis after Auricular Scratch in DM Mice Herpes simplex virus type 1 was inoculated on the right side in 210 mice, and 109 mice (51.9%) developed facial nerve paralysis on the inoculated side 6 to 8 days postinfection. The paralysis persisted for a week and recovered spontaneously in all cases. In a previous study, latent HSV-1 was detected from the geniculate ganglia of the mice that recovered from facial palsy.16 Ninety mice were selected randomly from the 109 mice and divided into three groups before the injection of STZ or citric acid. The induction of reactivation was performed using auricular scratching in 30 DM mice at 8 weeks after DM induction. Among these 30 mice, four had moderate facial nerve palsy on the right side 5 to 7 days after the reactivation induction (Table I). Five mice showed inactivity 2 to 5 days after the reactivation induction, and all of these mice died from encephalitis. Meanwhile, neither facial nerve paralysis nor death was observed in the DM mice without auricular scratching (n 5 30) or in the non-DM mice with auricular scratching (n 5 30). Laryngoscope 125: April 2015
Decrease of CD31/B2202 T Cells in the Spleen of DM Mice Cell-mediated immunity plays a major role in host defense against HSV-1.11 Therefore, we analyzed CD31 T cells in the spleens of mice with and without DM. Lymphocytes from the spleen were collected at 8 weeks after DM induction. The CD31/B2202 T-cell levels were significantly decreased in the DM mice compared with the non-DM mice (P < 0.01; Fig. 2A and 2B).
PCR Evaluation of Reactivated HSV-1 in the Facial Nerve The intratemporal portion of the facial nerve was resected and examined for HSV-1 DNA by PCR. Herpes simplex virus type 1 DNA was found in six of 15 DM mice after the reactivation induction, and two of these mice had facial nerve palsy on the right side (Table II). As shown in Figure 3, HSV-1 DNA was detected in two mice (4 and 5) with paralysis and in one mouse (1) without paralysis on the right side. Meanwhile, no HSV-1 DNA was detected in DM mice without auricular scratching or in non-DM mice with auricular scratching.
TABLE II. HSV-1 DNA from the Intratemporal Portion of the Facial Nerve. Mice
Auricular Scratch
Right (inoculated)
Left
DM
1
6/15 * (40.0%)
0/15 (0%)
DM
2
0/15
0/15
1
(0%) 0/15
(0%) 0/15
(0%)
(0%)
Non-DM
*P < 0.001 compared with the other groups. DM 5 diabetes mellitus.
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Fig. 3. Polymerase chain reaction analyses of the HSV-1 genomes from the bilateral facial nerves of DM mice at 10 days after auricular scratching (n 5 5). Three mice (1, 2, and 3) had no facial palsy, whereas two mice (4 and 5) had facial nerve palsy on the right side. The HSV-1 genome was detected in three mice (1, 4, and 5) on the right side. DM 5 diabetes mellitus; L 5 left facial nerve; N 5 negative control; P 5 positive control; R 5 right facial nerve; HSV-1 5 herpes simplex virus type 1.
Fig. 4. Electron microscopy images of the right intratemporal facial nerve and right geniculate ganglion at 10 days after auricular scratching. (a) Facial nerve of a DM mouse with paralysis after auricular scratching. The facial nerve was edematous, and myelin degeneration was observed. Some fibers were demyelinated with vascular changes in Schwann cells. (b) Facial nerve of a DM mouse without paralysis after auricular scratching, showing slight myelin degeneration. (c) Facial nerve of a non-DM mouse after auricular scratching, showing no myelin degeneration. (d) Geniculate ganglion of a DM mouse with palsy. Numerous HSV-1 capsids were observed in the rough endoplasmic reticulum. (e) Magnified image of the geniculate ganglion, showing HSV-1 capsids (arrows). Scale bars indicate 2 mm (a, b, and c), 500 nm (d), and 200 nm (e). DM 5 diabetes mellitus; GG 5 geniculate ganglion; HSV-1 5 herpes simplex virus type 1.
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Esaki et al.: Facial Nerve Palsy After Reactivation of HSV-1 in DM Mice
Electron Microscopic Examination of the Right Facial Nerve and Geniculate Ganglia Finally, we used transmission electron microscopy to examine the right facial nerve and geniculate ganglia of DM mice at 10 days after the induction of HSV-1 reactivation. The facial nerve of a DM mouse with paralysis was edematous and showed myelin degeneration. Some fibers were demyelinated with vascular changes in the Schwann cells (Fig. 4a). No fibers were demyelinated in the facial nerve of a DM mouse without paralysis, but slight myelin degeneration was observed (Fig. 4b). No myelin degeneration was observed in the facial nerve of a DM mouse without scratching (data not shown) or in that of a non-DM mouse with auricular scratching (Fig. 4c). The geniculate ganglia were examined, and numerous HSV-1 particles were observed in some ganglia, which had well-preserved fine structures, of a DM mouse with paralysis (Fig. 4d). Figure 4e shows a magnified image of the geniculate ganglia in which numerous HSV-1 particles can be observed in the rough endoplasmic reticulum.
DISCUSSION The incidence of DM is 10.5% in Japan, as reported by the Ministry of Health, Labor, and Welfare of Japan, whereas the incidence of DM in Bell’s palsy patients is more than 20%.17 This suggests that facial nerve paralysis develops more frequently in DM patients. Bell’s palsy is mainly caused by latent HSV-1 in the geniculate ganglion of the facial nerve.1,2 However, DM is closely related to cranial mononeuropathy and segmental motor paralysis.18–20 Thus, if DM patients develop facial nerve palsy, whether HSV-1 or DM is the cause of the facial palsy is unclear. This issue is important when we use steroids for medical treatment because steroid administration can exacerbate the conditions of DM patients. Neuropathy of the third cranial nerve is thought to be caused by diabetic mononeuropathy or angiopathy because it develops frequently in severe DM.21 Bell’s palsy is also more frequently observed in DM patients; however, it can develop regardless of the severity of DM, and the prognosis of facial nerve palsy does not differ between patients with and without DM.6 These differences suggest that the causes of the neuropathy of the third and seventh nerves could be different. Previously, we reported that facial paralysis induced by HSV-1 occurred more frequently in DM mice.10 Considering that DM amplifies the infectivity of HSV-1, we hypothesized that Bell’s palsy could easily be caused by the reactivation of HSV-1 under DM. In the present study, we investigated HSV-1 reactivation in DM mice and succeeded in inducing facial nerve paralysis. Mild skin trauma to the ear could induce HSV-1 reactivation in the murine trigeminal nerve.22 In contrast, skin trauma alone was not enough to induce HSV1 reactivation in the murine facial nerve. A previous study demonstrated that facial nerve paralysis could be induced after auricular scratching followed by the depletion of CD31 cells via an intraperitoneal injection of anti-CD3 monoclonal antibody.16 In DM patients, CD31, Laryngoscope 125: April 2015
CD41, CD81/CD282, and CD81/CD281 lymphocytes are significantly decreased compared with their levels in non-DM patients.13 Peripheral T lymphocytes were significantly decreased in patients with Bell’s palsy in the early stage of the disease.23,24 Both helper and cytotoxic T cells play major roles in host defense against HSV-111 and are thought to inhibit the spread of reactivated HSV-1. In the present study, recurrent facial nerve palsy could develop in DM mice after auricular scratching alone. After DM induction, CD31 cells decreased by 70%, which could accelerate development of facial nerve palsy after auricular scratching. The HSV-1 DNA was found in some of the DM mice without facial nerve paralysis and in the DM mice with paralysis. Electron microscopy also showed reactivated HSV-1 capsids in the geniculate ganglia of the DM mice with paralysis. Auricular scratching did induce HSV-1 reactivation in some DM mice, and facial nerve paralysis was observed in a subset of these DM mice with reactivated HSV-1 in the geniculate ganglia. A previous study also reported that the incidence of HSV-1 reactivation (67%) was more frequent than that of facial nerve paralysis (20%).16 Interestingly, the ratio of paralysis to HSV1 reactivation in the previous study (20%:67%) was similar to that in the current study (13%:40%). In the previous study, evoked electromyography remained at 60% to 70% when facial nerve palsy had recovered completely.10 We assume that the subclinical reactivation of HSV-1 disturbed the facial nerve to some extent but failed to develop into paralysis.
CONCLUSION We induced recurrent facial nerve paralysis in DM mice. The recurrent paralysis occurred in the DM mice only after auricular scratching. Auricular scratching and a decrease in CD31 T cells in the presence of DM induced the reactivation of HSV-1 in the geniculate ganglia, which resulted in facial nerve paralysis. Here, we propose that facial nerve paralysis in patients with DM arises as the result of HSV-1 reactivation, as is the case in Bell’s palsy, rather than as the result of a diabetic disorder.
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