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Quantitative histologic study of sural nerves in xeroderma pigmentosum
Case Reports
Quantitative Histologic Study of Sural Nerves in Xeroderma Pigmentosum Yoshihiro Origuchi, MD, lsematsu Eda, MD, Shin-ichi M a t s u m o t o , M D , a n d Akio Furuse, MD
The sural nerves of 2 siblings, 7 and 6 years of age with Group A xeroderma pigmentosum, were biopsied. The densities of myelinated fibers, 5,808/mm 2 and 5,163/mm 2, respectively, were strikingly decreased in comparison to control data. Both large and small myelinated fibers were reduced. Electron microscopy demonstrated many collagen fibers in the endoneurium and some collagen pockets. The loss of myelinated fibers was less severe than in previously reported patients. This discrepancy may be due to age differences at biopsy; our patients were biopsied at the ages of 7 and 6 years, while those patients reported previously were 10 years of age or older. The incidence of neurologic manifestations in xeroderma pigmentosum may increase after 6 years of age. Origuchi Y, Eda 1, Matsumoto S, Furuse A. Quantitative histologic study of sural nerves in xeroderma pigmentosum. Pediatr Neurol 1987;3:356-9.
of Group A xeroderma pigmentosum develop gradually, including areflexia, sensorineural hearing impairment, and mental retardation [3]. This study reports the quantitative histologic findings of the sural nerves of 2 siblings with Group A xeroderma pigmentosum which were biopsied at 7 and 6 years of age, respectively.
Methods Sural nerve biopsies were performed under intravenous anesthesia in the patients at the ages of 7 and 6 years, respectively. Each specimen was fixed in a solution of 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at room temperature, then was washed in 0.1 M cacodylate buffer for 24 hours, and finally was postfixed for 2 hours in a 2% solution of osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) [5]. After dehydration with an ascending alcohol series and propylene oxide, the specimens were embedded in Quetol-812. Transverse sections (1 ~tm) were cut with glass knives with a Reichert ultramicrotome, stained with 1% toluidine-blue, and then observed under a light microscope. Light photomicrographs were obtained according to the methods reported elsewhere [5] and the number and size distributions of myelinated fibers were determined by using a Kontron quantitative digital image analyzer [5]. For electron microscopy, ultrathin sections were cut with glass knives with a Reichert ultramicrotome, stained twice with uranyl acetate and lead citrate, and then studied with a Hitachi H-300 electron microscope.
Case Reports Patient I. This 8-year-old girl demonstrated sensitivity to sunlight upon initial exposure and developed a rash and pigmentation similar to freckles on her face at age 1 month. She was diagnosed as having xerodemla pigmentosum at the age of 8 months. She was admitted to our hospital at the age of 6 years. Her parents were healthy and had no history of consanguinity. Her delivery was uncomplicated and her developmental milestones were normal initially. She could walk at I year, 4 months of age and only rarely spoke a word by age 6 years. After entering kindergarten at age 6 years, she began to speak immediately. An intelligence test could not be administered because of lack of cooperation, Deep tendon reflexes were absent and Babinski signs were flexor bilaterally. Her hearing was normal. No corticospinal or cerebellar signs were detected. At 6 years, 9 months of age, motor nerve conduction velocity (MCV) was 50.7 m/sec in the right tibial nerve and sensory nerve conduction velocity (SCV) was not measurable in the right sural nerve. At 8 years, 9 months of age, MCV was 39.9 m/sec in the same nerve,
Introduction Xeroderma pigmentosum may be associated with many neurologic complications, such as microcephaly, mental deterioration, choreoathetosis, ataxia, spasticity, nerve deafness [1-3], and peripheral nerve disorders. Pathologic features of the peripheral nerve were studied in some reports [2,4]; however, quantitative histologic study in children 10 years of age or younger rarely has been reported. After 6 years of age, the major neurologic features
From the Department of Pediatrics: Nishibeppu National Hospital; Oita, Japan.
356
PEDIATRIC NEUROLOGY Vol. 3 No. 6
Patient 2. This boy is the younger brother of Patient 1. He had been delivered without complication and his developmental milestones initially were normal. He walked unassisted at age 12 months and could speak a few words at age 4 years. At 4 months of age he demonstrated sensitivity to sunlight upon initial exposure and suffered a sunburn reaction on his face; he developed the same pigmentation as his sister. The diagnosis of xeroderma pigmentosum was established. He was admitted to our hospital at 6 years of age. An intelligence test could not be administered because of lack of cooperation. All deep tendon reflexes were absent and Babinski signs were flexor bilaterally. His hearing was normal. No corticospinal or cerebellar signs were evident. At 5 years, 3 months of age MCV was 38.6 m/sec in the right tibial nerve and SCV
Communications should be addressed to: Dr. Origuchi; Department of Pediatrics; Nishibeppu National Hospital; 4548 Tsurumi, Beppu; Oita 874, Japan. Received August 25, 1987; accepted October 13, 1987.
Figure 1. Photomicrographs of transverse sections of the sural nerves, toluidine blue (x875); (A) Control patient at 10 years of age. (B) Patient 1 at 7 years of age. (C) Patient 2 at 6 years of age. There is marked loss of myelinated fibers from nerves of Patients I and 2.
was not measurable in the right sural nerve. At 7 years, 3 months of age MCV was 35.8 m/sec in the same nerve.
Results The most obvious abnormality revealed by light microscopy was a severe loss of myelinated fibers without evidence of myelin breakdown (Fig 1). Marked endoneurial fibrosis was observed. Figure 2 demonstrates histograms of myelinated fibers and depicts fiber density and areas of nerve fascicles. The peak of myelinated nerve fiber diameters was 2.0-3.0 ~tm, which was the same as the control, but myelinated fiber densities were strikingly reduced 2 2 to 5,808/mm and 5,163/ram . The loss ofmyelinated fibers was striking, but the surviving axons did not manifest any remarkable changes on electron microscopy (Fig 3A,B). Many collagen fibers were present in the endoneurium and some collagen pockets were observed. Rarely, small onionbulb formations were found (Fig 3A). No remarkable change was observed in the structure of unmyelinated fibers, but densities were 55,612/mm 2 and 2 46,234/mm , respecnvely, and were decreased compared to controls [5].
Discussion The critical age at which the major neurologic features developed gradually in Group A xeroderma pigrnentosum was 6 years [3]. The present patients were biopsied at 7 and 6 years of age, respectively. Fukuhara et al. [4] reported three patients, 11, 12, and 15 years of age, who had
peripheral nerve involvement most markedly in myelinated fibers; initially, large fibers were involved and later smaller ones became affected. In the present patients, the densities of both large and small myelinated fibers already were decreased, although the changes in the former were more distinct than in the latter. The loss of myelinated fibers was less severe than in patients reported previously [2,4]. This result may be due to age differences at biopsy; our patients were 7 and 6 years of age, while those patients reported previously were age 10 years or older. The incidence of neurologic manifestations in xeroderma pigmentosum may increase with age which is consistent with a chronic, progressive, degenerative neuronal disease [3]. The MCV of Patient 1 was 50.7 m/sec at 6 years, 9 months of age. Two years later it had decreased to 39.9 m/sec in the same nerve. The MCV of Patient 2 was 38.6 m/sec at the age of 5 years, 3 months, but had decreased to 35.8 m/sec in the same nerve 2 years later. This decrease implies that the major neurologic features develop in Group A xeroderma pigmentosum about the age of 6 or 7 years. Conversely, the SCVs of these 2 patients were not detected in the sural nerves at the ages of 6 years, 9 months, and 5 years, 3 months, respectively. This finding suggests that the sensory nerve is impaired earlier and more profoundly than the motor nerve in xeroderma pigmentosum. The exact mechanisms by which neurologic abnormalities develop in xeroderma pigmentosum remain unknown. Doxorubicin hydrochloride, known to interfere
Origuchi et al: XerodermaPigmentosum 357
4000
CONTROL
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M
lOy
S =0.407 mm"* /mm 2
200(] I000
S
400C
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CASE 1
8602
3000
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7y 7m
S = O. 349 mm 2 MF= 5 , 8 0 8 / m m 2
200G
I00C
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15p
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with RNA-DNA synthesis, has been reported to cause ganglioneuropathy [6] and loss of large myelinated fibers [7], as in the present patients. Patients with xeroderma pigmentosum have a defect in the repair of DNA damage induced by various substances [81, as well as by ultraviolet radiation. Based on their clinical and DNA repair studies of xeroderma pigmentosum, Robbins et al. 18] postulated that efficient DNA repair was required to maintain the integrity of neuronal DNA, thereby preventing premature deaths of neurons which may result from unrepaired DNA. In the human brain, DNA content increases rapidly for up to 10 months postnatally and reaches adult levels by 2 years of age. Mitoses no longer occur after 2 years of age. In xeroderma pigmentosum, many patients demonstrate sensivity to sunlight, developing rashes, pigmentation, and skin carcinoma. These skin disorders can be prevented by sheltering patients from ultraviolet exposure; however, neurologic abnormalities, such as areflexia or spasticity, cannot be prevented. This finding suggests that these neurologic changes may result from not only a defect in the repair of DNA damage, but also from other factors, though this has not been determined conclusively and requires further elucidation.
CASE 2
8603 M
6y4m
S = O.829 mrn 2 MF= 5,163 /ram 2
200(;
This study was supported in part by Grant No. 86-17 from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan and by a grant from the Neurocutaneous Syndrome Research Committee of the Ministry of Health and Welfare of Japan.
loo0
5
10
15p
Figure 2. Histograms of myelinated fibers. (A) Control patient. (B) Patient 1. (C) Patient 2. S = Areas of nerve fasicles; MF = Myelinated fiber density.
Figure 3. Electron micrographs of the sural nerve of Patient 2. (A) Onion-bulb formation concentrically arranged by overlapping of Schwann cell processes (xl 1,000). (B) Many collagen fibers and marked loss of myelinated fibers observed (x4,200).
358
PEDIATRIC NEUROLOGY Vol. 3 No. 6
References
[1] Hananian J, Cleaver JE. Xeroderma pigmentosum exhibiting neurological disorders and systemic lupus erythematosus. Clin Genet 1980; 17: 39-45. [2] Hakamada S, Watanabe K, Sobue G, Hara K, Miyazaki S. Xeroderma pigmentosum: Neurological, neurophysiological, and morphological studies. Eur Neurol 1982;21:69-76. [3] Mimaki T, Itoh N, Abe J, et al. Neurological manifestation in xeroderma pigmentosum. Ann Neurol 1986;20:70-5. [4] Fukuhara N, Kumamoto T, Takasawa H, Tsubaki T, Origuchi Y. The peripheral neuropathy in De Sanctis-Cacchione syndrome. Histological, ultrastructural, and morphomelric studies. Acta Neuropathol 1982;56:194-200.
[5] Origuchi Y. Quantitative histological study on the sural nerves of children. Brain Dev 1981;3:395-402. [6] Cho ES. Toxic effects of adriamycin on the ganglia of the peripheral nervous system: A neuropathological study. J Neuropathol Exp Neurol 1977;36:907-15. [7] Ohnishi A. Primary sensory neuron in experimental adriamycin intoxication. Neurol Med Chir 1979; 10:78-85. [8] Robbins H, Polinsky RJ, Moshell AN. Evidence that lack of deoxyribonucleic acid repair causes death of neurons in xeroderma pigmentosum. Ann Neurol 1983; 13:682-4.
Origuchi et al: Xeroderma Pigmentosum 359
Quantitative Histologic Study of Sural Nerves in Xeroderma Pigmentosum Yoshihiro Origuchi, MD, lsematsu Eda, MD, Shin-ichi M a t s u m o t o , M D , a n d Akio Furuse, MD
The sural nerves of 2 siblings, 7 and 6 years of age with Group A xeroderma pigmentosum, were biopsied. The densities of myelinated fibers, 5,808/mm 2 and 5,163/mm 2, respectively, were strikingly decreased in comparison to control data. Both large and small myelinated fibers were reduced. Electron microscopy demonstrated many collagen fibers in the endoneurium and some collagen pockets. The loss of myelinated fibers was less severe than in previously reported patients. This discrepancy may be due to age differences at biopsy; our patients were biopsied at the ages of 7 and 6 years, while those patients reported previously were 10 years of age or older. The incidence of neurologic manifestations in xeroderma pigmentosum may increase after 6 years of age. Origuchi Y, Eda 1, Matsumoto S, Furuse A. Quantitative histologic study of sural nerves in xeroderma pigmentosum. Pediatr Neurol 1987;3:356-9.
of Group A xeroderma pigmentosum develop gradually, including areflexia, sensorineural hearing impairment, and mental retardation [3]. This study reports the quantitative histologic findings of the sural nerves of 2 siblings with Group A xeroderma pigmentosum which were biopsied at 7 and 6 years of age, respectively.
Methods Sural nerve biopsies were performed under intravenous anesthesia in the patients at the ages of 7 and 6 years, respectively. Each specimen was fixed in a solution of 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at room temperature, then was washed in 0.1 M cacodylate buffer for 24 hours, and finally was postfixed for 2 hours in a 2% solution of osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) [5]. After dehydration with an ascending alcohol series and propylene oxide, the specimens were embedded in Quetol-812. Transverse sections (1 ~tm) were cut with glass knives with a Reichert ultramicrotome, stained with 1% toluidine-blue, and then observed under a light microscope. Light photomicrographs were obtained according to the methods reported elsewhere [5] and the number and size distributions of myelinated fibers were determined by using a Kontron quantitative digital image analyzer [5]. For electron microscopy, ultrathin sections were cut with glass knives with a Reichert ultramicrotome, stained twice with uranyl acetate and lead citrate, and then studied with a Hitachi H-300 electron microscope.
Case Reports Patient I. This 8-year-old girl demonstrated sensitivity to sunlight upon initial exposure and developed a rash and pigmentation similar to freckles on her face at age 1 month. She was diagnosed as having xerodemla pigmentosum at the age of 8 months. She was admitted to our hospital at the age of 6 years. Her parents were healthy and had no history of consanguinity. Her delivery was uncomplicated and her developmental milestones were normal initially. She could walk at I year, 4 months of age and only rarely spoke a word by age 6 years. After entering kindergarten at age 6 years, she began to speak immediately. An intelligence test could not be administered because of lack of cooperation, Deep tendon reflexes were absent and Babinski signs were flexor bilaterally. Her hearing was normal. No corticospinal or cerebellar signs were detected. At 6 years, 9 months of age, motor nerve conduction velocity (MCV) was 50.7 m/sec in the right tibial nerve and sensory nerve conduction velocity (SCV) was not measurable in the right sural nerve. At 8 years, 9 months of age, MCV was 39.9 m/sec in the same nerve,
Introduction Xeroderma pigmentosum may be associated with many neurologic complications, such as microcephaly, mental deterioration, choreoathetosis, ataxia, spasticity, nerve deafness [1-3], and peripheral nerve disorders. Pathologic features of the peripheral nerve were studied in some reports [2,4]; however, quantitative histologic study in children 10 years of age or younger rarely has been reported. After 6 years of age, the major neurologic features
From the Department of Pediatrics: Nishibeppu National Hospital; Oita, Japan.
356
PEDIATRIC NEUROLOGY Vol. 3 No. 6
Patient 2. This boy is the younger brother of Patient 1. He had been delivered without complication and his developmental milestones initially were normal. He walked unassisted at age 12 months and could speak a few words at age 4 years. At 4 months of age he demonstrated sensitivity to sunlight upon initial exposure and suffered a sunburn reaction on his face; he developed the same pigmentation as his sister. The diagnosis of xeroderma pigmentosum was established. He was admitted to our hospital at 6 years of age. An intelligence test could not be administered because of lack of cooperation. All deep tendon reflexes were absent and Babinski signs were flexor bilaterally. His hearing was normal. No corticospinal or cerebellar signs were evident. At 5 years, 3 months of age MCV was 38.6 m/sec in the right tibial nerve and SCV
Communications should be addressed to: Dr. Origuchi; Department of Pediatrics; Nishibeppu National Hospital; 4548 Tsurumi, Beppu; Oita 874, Japan. Received August 25, 1987; accepted October 13, 1987.
Figure 1. Photomicrographs of transverse sections of the sural nerves, toluidine blue (x875); (A) Control patient at 10 years of age. (B) Patient 1 at 7 years of age. (C) Patient 2 at 6 years of age. There is marked loss of myelinated fibers from nerves of Patients I and 2.
was not measurable in the right sural nerve. At 7 years, 3 months of age MCV was 35.8 m/sec in the same nerve.
Results The most obvious abnormality revealed by light microscopy was a severe loss of myelinated fibers without evidence of myelin breakdown (Fig 1). Marked endoneurial fibrosis was observed. Figure 2 demonstrates histograms of myelinated fibers and depicts fiber density and areas of nerve fascicles. The peak of myelinated nerve fiber diameters was 2.0-3.0 ~tm, which was the same as the control, but myelinated fiber densities were strikingly reduced 2 2 to 5,808/mm and 5,163/ram . The loss ofmyelinated fibers was striking, but the surviving axons did not manifest any remarkable changes on electron microscopy (Fig 3A,B). Many collagen fibers were present in the endoneurium and some collagen pockets were observed. Rarely, small onionbulb formations were found (Fig 3A). No remarkable change was observed in the structure of unmyelinated fibers, but densities were 55,612/mm 2 and 2 46,234/mm , respecnvely, and were decreased compared to controls [5].
Discussion The critical age at which the major neurologic features developed gradually in Group A xeroderma pigrnentosum was 6 years [3]. The present patients were biopsied at 7 and 6 years of age, respectively. Fukuhara et al. [4] reported three patients, 11, 12, and 15 years of age, who had
peripheral nerve involvement most markedly in myelinated fibers; initially, large fibers were involved and later smaller ones became affected. In the present patients, the densities of both large and small myelinated fibers already were decreased, although the changes in the former were more distinct than in the latter. The loss of myelinated fibers was less severe than in patients reported previously [2,4]. This result may be due to age differences at biopsy; our patients were 7 and 6 years of age, while those patients reported previously were age 10 years or older. The incidence of neurologic manifestations in xeroderma pigmentosum may increase with age which is consistent with a chronic, progressive, degenerative neuronal disease [3]. The MCV of Patient 1 was 50.7 m/sec at 6 years, 9 months of age. Two years later it had decreased to 39.9 m/sec in the same nerve. The MCV of Patient 2 was 38.6 m/sec at the age of 5 years, 3 months, but had decreased to 35.8 m/sec in the same nerve 2 years later. This decrease implies that the major neurologic features develop in Group A xeroderma pigmentosum about the age of 6 or 7 years. Conversely, the SCVs of these 2 patients were not detected in the sural nerves at the ages of 6 years, 9 months, and 5 years, 3 months, respectively. This finding suggests that the sensory nerve is impaired earlier and more profoundly than the motor nerve in xeroderma pigmentosum. The exact mechanisms by which neurologic abnormalities develop in xeroderma pigmentosum remain unknown. Doxorubicin hydrochloride, known to interfere
Origuchi et al: XerodermaPigmentosum 357
4000
CONTROL
8103
300O
M
lOy
S =0.407 mm"* /mm 2
200(] I000
S
400C
1~p
tO
CASE 1
8602
3000
F
7y 7m
S = O. 349 mm 2 MF= 5 , 8 0 8 / m m 2
200G
I00C
S
~b
15p
4O(;O
with RNA-DNA synthesis, has been reported to cause ganglioneuropathy [6] and loss of large myelinated fibers [7], as in the present patients. Patients with xeroderma pigmentosum have a defect in the repair of DNA damage induced by various substances [81, as well as by ultraviolet radiation. Based on their clinical and DNA repair studies of xeroderma pigmentosum, Robbins et al. 18] postulated that efficient DNA repair was required to maintain the integrity of neuronal DNA, thereby preventing premature deaths of neurons which may result from unrepaired DNA. In the human brain, DNA content increases rapidly for up to 10 months postnatally and reaches adult levels by 2 years of age. Mitoses no longer occur after 2 years of age. In xeroderma pigmentosum, many patients demonstrate sensivity to sunlight, developing rashes, pigmentation, and skin carcinoma. These skin disorders can be prevented by sheltering patients from ultraviolet exposure; however, neurologic abnormalities, such as areflexia or spasticity, cannot be prevented. This finding suggests that these neurologic changes may result from not only a defect in the repair of DNA damage, but also from other factors, though this has not been determined conclusively and requires further elucidation.
CASE 2
8603 M
6y4m
S = O.829 mrn 2 MF= 5,163 /ram 2
200(;
This study was supported in part by Grant No. 86-17 from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan and by a grant from the Neurocutaneous Syndrome Research Committee of the Ministry of Health and Welfare of Japan.
loo0
5
10
15p
Figure 2. Histograms of myelinated fibers. (A) Control patient. (B) Patient 1. (C) Patient 2. S = Areas of nerve fasicles; MF = Myelinated fiber density.
Figure 3. Electron micrographs of the sural nerve of Patient 2. (A) Onion-bulb formation concentrically arranged by overlapping of Schwann cell processes (xl 1,000). (B) Many collagen fibers and marked loss of myelinated fibers observed (x4,200).
358
PEDIATRIC NEUROLOGY Vol. 3 No. 6
References
[1] Hananian J, Cleaver JE. Xeroderma pigmentosum exhibiting neurological disorders and systemic lupus erythematosus. Clin Genet 1980; 17: 39-45. [2] Hakamada S, Watanabe K, Sobue G, Hara K, Miyazaki S. Xeroderma pigmentosum: Neurological, neurophysiological, and morphological studies. Eur Neurol 1982;21:69-76. [3] Mimaki T, Itoh N, Abe J, et al. Neurological manifestation in xeroderma pigmentosum. Ann Neurol 1986;20:70-5. [4] Fukuhara N, Kumamoto T, Takasawa H, Tsubaki T, Origuchi Y. The peripheral neuropathy in De Sanctis-Cacchione syndrome. Histological, ultrastructural, and morphomelric studies. Acta Neuropathol 1982;56:194-200.
[5] Origuchi Y. Quantitative histological study on the sural nerves of children. Brain Dev 1981;3:395-402. [6] Cho ES. Toxic effects of adriamycin on the ganglia of the peripheral nervous system: A neuropathological study. J Neuropathol Exp Neurol 1977;36:907-15. [7] Ohnishi A. Primary sensory neuron in experimental adriamycin intoxication. Neurol Med Chir 1979; 10:78-85. [8] Robbins H, Polinsky RJ, Moshell AN. Evidence that lack of deoxyribonucleic acid repair causes death of neurons in xeroderma pigmentosum. Ann Neurol 1983; 13:682-4.
Origuchi et al: Xeroderma Pigmentosum 359