Brain morphology in Duchenne muscular dystrophy: A Golgi study

Original Articles

Brain Morphology in Duchenne Muscular Dystrophy: A Golgi Study Venkita Jagadha, MD and Laurence E. Becker, MD

Intellectual impairment associated with Duchenne muscular dystrophy is well recognized, although no consistent anatomic central nervous system lesions have been reported. The autopsy findings of 13 patients, ages 1318 years, were reviewed. Intelligence quotients, ascertained in 5 patients, ranged from 46-79. Gross and microscopic examinations of brain and spinal cord revealed no consistent pattern of abnormalities. Neuropathology included neuronal loss and gliosis in spinal gray matter and tegmental brainstem, extensive Purkinje cell loss, mononuclear perivascular cuffing with cortical and subcortical gliosis, and cerebral heterotopia. Quantitative analysis of rapid Golgi impregnations of the visual cortex revealed significantly reduced dendritic length and branching of apical and basal dendrites from pyramidal neurons in 1 patient and less striking attenuation of dendritic arborization in 2 others. The literature suggests that the intellectual deficit in Duchenne muscular dystrophy is nonprogressive and unrelated to age or severity of muscle disease, although performance intelligence quotient may deteriorate with progressive muscle weakness. Golgi analysis suggested that abnormal dendritic development and arborization may underlie intellectual impairment. Although the pathogenesis of the cellular defect is not fully known, the coexistence of central nervous system and muscle pathology raises the possibility of a common molecular mechanism. Jagadha V, Becker LE. Brain morphology in Duchenne muscular dystrophy: A Golgi study. Pediatr Neurol 1988; 4:87-92.

Introduction The involvement of the central nervous system (CNS) in Duchenne muscular dystrophy (DMD) is well recognized. Earlier studies attempted to ascribe mental retardation in these patients to the effects of the physical handicap [1-3], but numerous well-documented reports indicated that decreased intellectual function may be an integral part of

From the Division of Neuropathology (Department of Pathology); The Hospital for Sick Children; Toronto, Ontario, Canada.

the disease [4-21]. In patients with DMD the mean IQ is about 85; the distribution appears normal, although it has an overall skew toward the low range [4,5,10,12,15,19]. Kozicka et al. found IQ values lower than 84 in 60% of patients with DMD [ 14]. Most of these patients were in the group with mild mental retardation (IQ 52-57) or in the borderline normal group (IQ 68-83). In this X-linked recessive disorder, muscle involvement begins early, as reflected by elevated serum creatine kinase activities at birth. Thus, it is reasonable to assume that CNS involvement may also occur early in life. We conducted a retrospective study of 13 DMD patients, assessed at The Hospital for Sick Children, who had complete autopsies performed. Golgi studies were performed in 3 patients to delineate the patterns of dendritic arborization.

Methods Thirteen patients who had a clinical diagnosis of DMD confirmed by biopsy and who had undergone a complete postmortem examination were selected for the study. The patients ranged in age from 13-18 years at the time of death. The clinical data were reviewed in detail, particularly with regard to psychometric assessment. The micro~opic sections of various proximal and distal skeletal muscle groups, other viscera, and sections of the brain and spinal cord were reviewed. In 3 patients, material was obtained for Golgi studies from the visual cortex (area OC of von Economo). This site was originally chosen for morphometric dendritic assessments because of the possibility of correlating a morphologic parameter (dendritic alteration) and a clinical parameter (e.g., visual evoked responses). The visual cortex is readily identified in immature brains because it borders the calcarine sulcus; thus, consistency of sampling is assured. Tissue for Golgi studies was obtained only in cases in which autopsies were performed within 24 hours of death (3 patients). A 5 mm slice was cut from the unfixed brain and processed by the modified rapid Golgi technique [22,23]. The specimens were blocked in paraffin and sections cut at 50 lam intervals. Camera lucida drawings (magnification: x270) of individual layer 3 and layer 5 pyramidal neurons were used to trace the dendritic morphology. For quantitative studies of dendritic arborization, the following parameters were assessed: total dendritic length, number of branch orders, number of branches, branch length by order, and number of dendritic intersections. Ten fully impregnated layer 3 and layer 5 neurons were studied. The number of dendritic intersections was assessed by Scholl's concentric circle method [22,231 in which a grid with concentric circles, each 20 ~tm apart, is placed with its center corresponding to the cell body. As they cross the concentric circles,

Communications should be addressed to: Dr. Becker; Division of Neuropathology: The Hospital for Sick Children: 555 University Avenue: Toronto, Ontario, Canada M5G IX8. Received October 27, 1987; accepted February 29, 1988.

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Table 1.

Pathologic findings in 13 patients with Duchenne muscular dystrophy

Age at Death (years~

IQ

Brain Weight (grams)

1

15

NA

1,383

Neuronal loss and moderate astrogliosis in spinal gray matter and tegmental brainstem

2

16

NA

1,440

No significant pathology

3

14

NA

1,330

No significant pathology

4

15

NA

1,432

No significant pathology

5

15

50

1,176

Chronic encephalitis with widespread neuronal loss, gliosis, and perivascular mononuclear cuffs

6

18

NA

1,268

No significant pathology

7

16

NA

1,359

No significant pathology

8

17

NA

1,300

No significant pathology

9

15

NA

1,400

Neuronal heterotopia in periventricular white matter

10

15

Dullnormal

1,575

No significant pathology

11

16

Dullnormal

1,350

No significant pathology

12

16

Verbal: 79, performance: 97

1,620

No significant pathology

13

13

46

1,300

Recent hypoxic-ischemic damage

Patient Number

Neuropathologic Findings

Abbreviation: NA = Not available dendritic branches are counted and the numbers plotted as a function of the distance from the cell body. For branch orders, a centrifugal ordering system was used [22,23]. Assessment of the number of branches and branch orders, brunch length by order, and total dendritic length was made by placing a camera iucida drawing on an Apple II* graphic tablet interfaced with a programmed computer. All the parameters used in quantitative analysis were compared with those of an age-matched, neurologically normal, 16-yeur-old control male. The Student t test was applied to determine the statistical significance of differences between values from DMD patients and the control.

patients were within normal limits (Table 1). Microscopic pathology, observed in 4 patients, included mild neuronal loss and moderate astrogliosis in the anterior horn cell column of the spinal gray matter and tegmental brainstem (Patient 1), extensive Purkinje cell loss and mononuclear perivascular cuffing with gliosis in the cortical and subcortical areas (Patient 5), a small focus of neuronal heterotopia in the cerebral white matter (Patient 9), and recent hypoxicischemic damage (Patient 13).

Results The available psychometric assessments are presented in Table 1. Samples of various proximal and distal muscle groups demonstrated a severe dystrophic process with replacement of the muscle by fibrofatty tissue in most patients. Active necrosis was identified focally in Patient 12, while the pathology was consistent with end-stage disease in other patients. No abnormalities were found on gross examination of the brain and spinal cord in these patients. The brain weight of Patient 5 was 1,176 gm, while the brain weights of the other

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Quantitative Golgi Studies

Total Dendritic Length. In layer 3, total dendritic length of both apical and basal dendrites in Patient 12 was significantly reduced when compared with the control brain. In layer 5, the length of both apical and basal dendrites was significantly reduced in Patient 12; in Patient 11, only basal dendrites were significantly reduced (Table 2). Number of Branch Orders. In layer 3, the number of branch orders of both apical and basal dendrites was decreased significantly (p < 0.01) in Patient 12. In layer 5,

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Table 3.

Number of intersections of dendrites at 20 ~tm intervals up to 300 gm from the cell body in DMD and an age-matched control Layer 3 Patients

Control

Mean

SD

Pt No.

Mean

SD

Difference (p value)

43.1

9.90

11

27.5

1 t.57

< 0.01

< 0.05

12

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741

< 0.001

15.36

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13

43.2

22.92

NS

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29.46

NS

11

34.4

18.51

NS*

12

28.9

13.84

< 0.05

12

29.8

9.32

13

53.6

27.01

NS

13

46.1

18.86

Type of Dendrite

Mean

SD

Pt No.

Mean

Apical

36.6

9.01

11

39.6

8.87

NS

12

24.7

7.53

13

43.6

11

Basal

45.7

13.79

Layer 5 Patients

Control Difference {p value)

SD

41.3

7.75

< 0.01 NS

NS = Not significant * Although there was no statistically significant difference in the total number of (lower and lateral) intersections, a significant diminution was observed in the lower quadrant (19< 0.01 ).

the number of both apical and basal dendrites also was significantly reduced (p < 0.01) in Patient 12. The number of apical dendrites in Patient 11 was even more significantly reduced (p < 0.001). All other differences were not significant. Number of Branches. In layer 3, the number of branches of apical dendrites in Patient 12 was significantly decreased; in Patient 13, basal dendrites were significantly decreased (Table 2). In layer 5, only the number of apical dendrites was significantly decreased in Patients 11 and 12. Branch Length by Order. In Patients 11 and 12, branch length of layer 3 apical dendrites was significantly reduced in orders 5 to 8. Number of Dendritic Intersections. The number of layer 3 intersections was significantly reduced in Patient 12,

while the number of layer 5 intersections was significantly reduced in both Patients 11 and 12 (Table 3, Fig 1).

Discussion Significant impairment of intellectual function must be considered an integral part of DMD. Most authors report that intellectual retardation does not appear to worsen with time [5,10,12,19], which is in contrast to the progressive nature of the muscle disease. In most studies no positive correlation has been determined between the IQ values and the age of the patient or the severity and duration of the disease [4,5,10] although Rosman [13] described a direct relationship between severity of clinical disease, severity of pathologic involvement, and impairment of intellectual function.

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200

240

280

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DISTANCE FROM CELL BODY (p.m) Figure 1. Dendritic intersections in apical dendrites o f layer 5 neurons." C) = control, • = Patient 1l, • = Patient 12, 0 = Patient 13.

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Early verbal disability, reflected by a low verbal IQ, is said to be characteristic of DMD [1538]. In some patients, nonverbal intellectual skills may be impaired as well [15,18]. Marsh and Munsat found that the mildly affected group scored lower on the verbal than the performance scale, while the more disabled group demonstrated no significant differences between the two scores [15]. The decrease in verbal IQ cannot be attributed to the physical handicap; children with other neuromuscular disorders, such as infantile and juvenile muscular atrophy, who have more marked muscle weakness, have normal IQ values [ 17]. Occasionally, intellectual retardation may precede the onset of clinically evident muscle weakness in children with DMD [7]. Vignos compared the IQ values of DMD patients with those of their siblings and found values of 85 and 110, respectively [17]. Prosser et al. found a similar difference between the IQ values of patients and unaffected siblings [12]; however, the mean IQs of both patient and siblings in a family in the upper income bracket were higher than those of children in the lower income groups [ 12]. Mental impairment is no more severe in sporadic than familial cases [16]. Rosman and Kakulas described several morphologic changes in the brains of patients with DMD [9]. They studied 12 patients, 6 of whom had documented mental deficiency (3 with DMD, 3 with myotonic dystrophy). The other 6 patients were used as controls, 4 of whom had DMD. Three of the 4 control patients died at 26, 31, and 44 years of age, suggesting that a milder form of dystrophy may have been present. They concluded that the mean brain weight in the mentally defective group was lower than in the control group. The mentally defective patients had abnormally globular brains, as well as anteroposterior foreshortening (3 patients), pachygyria (4 patients), and microscopic changes in the form of neuronal heterotopia (6 patients). Rosman reported 10 patients with DMD and described 1 patient with an abnormally globular brain, showing disordered cortical lamination [ 13]. Kakulas and Jacobsen compared the ratios of gray and white matter and also total parenchyma and ventricular size of 18 patients who had DMD with those of age-matched controls [21]. They concluded that the cortical ribbon was reduced and the gray matter was diminished relative to the white matter. These observations by Rosman and Kakulas [9], Rosman [13], and Kakulas and Jacobsen [21] were not confirmed in the present study or by other investigators [ 11]. We could not identify any consistent or characteristic pattern of gross or microscopic abnormalities in the brains of the present 13 patients with DMD. With the exception of 1 patient (Patient 5), all brain weights were within the normal range. This patient, who had an IQ of 50, had chronic encephalitis of undefined etiology, presumably coincidental. Because the findings of mild neuronal loss and astrogliosis of the spinal gray matter and tegmental brainstem in Patient 1 could not be corroborated in the other 12 patients, its relevance to the dystrophic process is questionable. A minute neuronal heterotopia was present in one other

patient. No definitive changes of cortical lamination or gyration were evident in any of these 13 patients. Our findings are consistent with those of Dubowitz and Crome [11]. Of their 21 patients with DMD, only 1 had low brain weight and no consistent gross or histologic findings were identified. Schmidt et al. recently described increased head circumference in patients with DMD, although no abnormalities were demonstrated on cranial computed tomography [24]. Even though conventional morphologic studies revealed only minimal pathology, abnormalities were found in the dendritic arborizational patterns in the 3 patients in whom quantitative Golgi analyses were undertaken. The most compelling evidence for dendritic abnormalities in this study comes from Patient 12 (verbal IQ 79) who demonstrated a very significant decrease in total dendritic length, as well as various branching parameters. These parameters included a number of dendritic intersections, branch orders, and branches involving both layer 3 and layer 5 neurons. In Patient 11, whose IQ was in the dull normal range, there was decreased dendritic arborization in layer 5 cells with a diminution in the total length of basal dendrites. The third patient (Patient 13 with evidence of terminal hypoxic damage; IQ 46), demonstrated relatively minor abnormalities, with a decreased number of branches of basal dendrites in layer 3 neurons. Thus, the attenuation of dendritic branching was not directly correlated to the IQ measurements in these patients. Because intellectual impairment in our patients could not be localized to a particular anatomic area, we assumed that dendritic changes are generalized (i.e., the alterations observed in the visual cortex also may occur elsewhere, accounting indirectly for reduced mental functioning). The observation that the impairment of intellectual function in DMD is nonprogressive suggests that the CNS may be involved at an early stage of development. In this Xlinked disorder the nature of the gene product remains unknown, but the most favored hypothesis points to an intrinsic defect of cell membranes. Recently, Infante suggested that an impaired synthesis of highly unsaturated phosphatidylcholines of the sarcoplasmic reticulum may be the primary defect in DMD [25]. Abnormalities of erythrocyte membranes and leukocyte function also have been found [26, 27]. These preliminary data suggest that the defect in DMD may be expressed widely in tissues other than muscle. Although the precise physiologic mechanisms are not established, dendritic abnormalities have been described in a variety of conditions associated with mental retardation [28-34]. These abnormalities range from arborization to anomalous morphology of spines. Changes in the number, length, and spatial arrangement of dendrites and dendritic spines have been described in conditions such as Down syndrome [31-33] (e.g., dendritic atrophy observed during early childhood in Down syndrome [33]). Dendritic abnormalities are also prominent features in a number of metabolic disorders, including gangliosidoses [34].

Jagadha and Becker: Brain Morphology in DMD

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Even though it was limited by the number of patients, our study suggests that abnormal dendritic development and arborization may be part of the pathologic spectrum in DMD. Further correlative studies with a larger series of patients may elucidate the anatomic substrate of mental retardation in DMD.

References

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