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Neuropathological investigation of patients with prolonged anorexia nervosa

Objectives Recent neuroimaging studies have indicated that the mesolimbic pathway, known to work as reward neuronal circuitry, regulates cognitive–behavioral flexibility in prolonged anorexia nervosa (AN). Although AN is associated with the highest mortality rate among psychiatric disorders, there have been few neuropathological studies on this topic. This study aims to identify alterations of the reward circuitry regions, especially in the nucleus accumbens (NAcc), using AN brain tissues. Methods The neuronal networks in AN cases and controls were examined by immunohistochemistry directed at tyrosine hydroxylase (TH; dopaminergic neuron marker) and glial fibrillary acidic protein (GFAP; astrocyte marker). We also immunochemically analyzed frozen samples presenting astrogliosis, especially in the NAcc and striatum. Results Histologically, neuronal deformation with cytoplasmic shrinkage was seen in reward‐related brain regions, such as the orbitofrontal cortex/anterior cingulate cortex. The NAcc showed massive GFAP‐positive astrocytes and dot‐like protrusions of astrocytes in the shell compartment. In the shell, TH and GFAP immunoreactivities revealed prominent astrogliosis within striosomes, which receive projection from the ventral tegmental area (VTA). The numbers of GFAP‐positive astrocytes in the NAcc (P = 0.0079) and VTA (P = 0.0025) of AN cases were significantly higher than those of controls. Strongly immunoreactive 18 to 25 kDa bands, which might represent degradation products, were detected only in the NAcc of AN cases. Clinically, all cases presented cognitive rigidity, which might reflect a deficit of the reward pathway. Conclusion Our findings suggest impaired dopaminergic innervation between the NAcc and VTA in AN. Functional dysconnectivity in the reward‐related network might induce neuropsychiatric symptoms associated with AN.

Anorexia nervosa (AN) is a serious psychosomatic eating disorder characterized by severe disturbances in eating behavior, body weight, and body/self-perception. 1 It sometimes shows familial groupings, and shared biological underpinnings have been hypothesized. 2 The most obvious features of AN are severe emaciation and malnourishment. 3 Approximately half of the cases are chronic or recurrent, with an average medical history of more than 5 years. In patients with AN, chronic starvation can cause medical and metabolic complications affecting virtually any body system, 3 and is associated with depressed mood, anxiety, obsessions, compulsions, and affective dysregulation. 4 AN is classified into two types on the basis of eating-related behavior, ie, restricting type (loss of weight is caused purely by dieting, without binge eating or purging) and binge eating/purging type (involving restricted food intake, but also periodic disinhibition of restraint, with binge eating and/or purging). 1,5 Transition between these syndromes frequently occurs.
The precise pathophysiology of AN remains unclear and might involve complex interactions of developmental, genetic, environmental, psychological, and neurobiological factors. Recent structural and functional magnetic resonance imaging (fMRI) studies have focused on the role of neurocircuitry in the pathophysiology of AN, and have implicated the reward circuit that regulates food intake. 3,5,6 The neurotransmitter dopamine modulates food reward via the mesocorticolimbic pathway of the brain, which includes the orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), hippocampus, ventral striatum/nucleus accumbens (NAcc), and midbrain/ventral tegmental area. 7,8 Those fMRI studies indicated the mesocorticolimbic pathway to be particularly vulnerable in earlier cases of AN. 5,9,10 Dysfunction may lead to emotional processing deficits and psychiatric symptoms such as obsessivecompulsive disorder, which then drive maladaptive behaviors in AN. 3,11 Although AN has the highest mortality rate of any psychiatric disorder, there has been been few neuropathological investigations. 12,13 In this study, we identified five autopy cases with prolonged AN in our archives. Considering the pathophysiological background, we planned to focus on the NAcc, which is a center of the mesocorticolimbic pathway. We evaluated the neuropathology of AN brains in comparison with control brains to identify alterations of the reward circuitry regions, especially in the NAcc and ventral tegmental area (VTA). Tyrosine hydroxylase (TH), calbindin, and choline O-acetyltransferase (ChAT) were utilized to identify anatomical subdivisions of the NAcc. In addition, we histochemically analyzed frozen samples, and considered the correlation between neuronal dysfunction and clinical symptoms.

Participants
Based on the reported criteria for AN, 1 we retrospectively identified five cases with binge-eating/purging-type AN in the autopsyconfirmed archives of the Department of Psychiatry; Tokyo Metropolitan Matsuzawa Hospital; the Dementia Research Project/Tokyo Metropolitan Institute of Medical Sciences; Nagoya Psychiatric Brain Bank Consortium; and the Institute for Medical Science of Aging, Aichi Medical University. Ten individuals without psychiatric or neurodegenerative disease were taken from our archives as controls (Supplementary Table S1).
For double-label immunofluorescence detection, brain sections were pretreated as described above and incubated overnight at 4 C with a cocktail of appropriate primary antibodies. The sections were washed and incubated with a cocktail of Alexa568conjugated goat anti-rabbit IgG (1:500, Vector Laboratories) and Alexa488-conjugated goat anti-mouse IgG (1:500, Vector Laboratories). After further washing, the sections were coverslipped with nonfluorescent mounting media (VECTASHIELD; Vector Laboratories) and observed with a BZ-X710 microscope (Keyence). Counts of immunopositive cells were undertaken in 7 to 9 visual fields per case that were randomly chosen from the NAcc (shell compartment) and VTA. All images were captured with the BZ-X710 microscope using the same settings. The areas of GFAP-positive cells in the NAcc were extracted and quantified by BZ-H3C Hybrid Cell Count Software (Keyence). For semiquantitative analyses of immunohistochemically stained tissue sections, the density of GFAP-positive astrocytes was graded 0 for absent, AE for low, + for intermediate, ++ for high, and +++ for severe, based on microscopic observations at Â200 magnification.

Statistical analysis
We compared the quantitative values of GFAP-positive astrocytes in the NAcc and VTA of AN and control cases using GraphPad Prism 7 software. The statistical significance of differences was assessed by application of the Mann-Whitney U test for nonparametric variables. The criterion of significant difference was set at P < 0.05. The average brain weight of AN cases was 1,209 AE 59.3g, which is within the range expected for the autopsied brain (before fixation) of an adult woman (1200-1350 g). 19 The case data are summarized in Table 1. Depressed mood was seen in 80% (four cases), anxiety in 60% (three cases), affective dysregulation in 60% (three cases), obsessions/compulsions in 40% (two cases), and cognitive rigidity in 100% (five cases) of AN cases. Computed tomography scan images were obtained in one case with AN (case #5) during hospitalization, and showed widespread sulcal enlargement and marked ventricular dilation (Fig. 1).  General neuropathological findings in AN cases There was no apparent regional atrophy in the neocortex, basal ganglia, hippocampus, midbrain, pons, or spinal cord in cases with AN ( Fig. 2a,b). Regarding neurodegenerative changes, neurofibrillary tangles (NFTs)/Aβ in the hippocampal and other regions showed Braak&Braak NFT stage 0 and Aβ stage 0 in all cases. No other aging-related proteins, including α-synuclein and TAR DNA-binding protein 43 (TDP-43), were detected ( Table 2).

Demographic data
Increase of astrocytes in reward-related brain regions Microscopically, severe astrogliosis with neuronal loss was prominent in the NAcc of all AN cases (Fig. 2c-e), in comparison with the NAcc in controls (Fig. 2f). Capillary blood vessels in the NAcc showed swelling of capillary endothelial cells, giving the appearance of venous collagenosis (Fig. 2d). In the striatum, moderate subependymal gliosis was present in the caudate nucleus. The OFC and ACC showed ischemic changes in neurons with a slight increase of astrocytes (Fig. 2g,h). Two cases showed a slight increase of astrocytes in hippocampus CA1 and subiculum (Fig. 2i,j). A slight increase of astrocytes was seen in the midbrain, including the SN and VTA (Fig. 2k,l). Some cases presented atrophic neurons with free melanin in the SN. There was a slight increase of astrocytes in the mediodorsal thalamus. The distribution of increased astrocytes is summarized in Table 2.
Immunoreactivity in the nucleus accumbens Astrogliosis, which was detected with anti-GFAP immunohistochemistry, was prominent in the shell (outer) compartment of the NAcc in all of cases, although the severity varied from case to case (Fig. 3a). In addition, many positive dot-like protrusions were stained for GFAP. In the shell compartment of the NAcc, intense TH-immunostaining was observed, in accordance with previous findings. 20 Alternate sections of the NAcc shell were stained for TH (Fig. 3b) and GFAP (Fig. 3c), and the area showed patchy GFAP immunoreactivity. GFAP-positive astrocytes were seen preferentially in striosomes with relatively light TH staining.
Regarding activated microglial cells, there were mild increases of CD68/Iba1-positive microglial cells in both the shell and core NAcc (Fig. 3a). The distribution of microglial marker immunoreactivity in the shell compartment of the NAcc was not patchy.
TH/GFAP double immunofluorescence revealed that the astrocyte burden differed between striosome and matrix of the NAcc in all AN cases (Fig. 3d,e). AN cases displayed prominent astrogliosis in striosomes identified on the basis of low TH immunostaining (Fig. 3d). Controls showed sparse GFAP-positive astrocytes in the striosomes or matrix in the NAcc (Fig. 3e).

Immunoreactivity in midbrain
Cases with AN showed mild to moderate astrogliosis in the SN (Fig. 4a) and VTA (Fig. 4b), although neuronal loss was not obvious.
Many positive dot-like protrusions were also stained for GFAP in the midbrain of AN cases. The SN in controls presented slight astrogliosis that could have occurred as a result of normal aging.

Quantifications of GFAP-positive astrocytes
The number of GFAP-positive astrocytes in the NAcc of AN cases was significantly higher than that of controls (Mann-Whitney U test, P = 0.0079) (Fig. 5a). In the SN, there was no significant difference between AN and controls (P = 0.3290), whereas the number of GFAP-immunopositive astrocytes in the VTA of AN cases was significantly higher than in controls (P = 0.0025) (Fig. 5b).

Immunoblot analysis
Anti-actin antibody staining confirmed that approximately equal quantities of Tris-soluble fraction were applied in each case. Anti-GFAP antibody revealed 5 to 6 bands of 40 to 50 kDa corresponding to GFAP in the posterior striatum and NAcc of AN case #5 and controls. Interestingly, strongly immunoreactive bands of ≈18 to 25 kDa, which may represent degradation products, were detected only in the NAcc of AN cases, suggesting that abnormal GFAP degradation may occur in this region (Fig. 6).

Discussion
Dopaminergic neuronal network in AN Anatomically, the NAcc is located in the caudal part of the anterior horn of the lateral ventricle, lying between the head of the caudate nucleus and the putamen, which forms the ventral striatum with olfactory tubercles. The NAcc can be subdivided into inner and outer parts (core and shell), and the striatum itself can be further subdivided into striosome and matrix, in addition to dorsal and ventral parts. 18 The shell is considered to be more important than the core for drug-reward trigger zones and receives strong projections from the VTA. 21 In this study, we found massive GFAP immunopositivity of astrocytes in the shell of the NAcc, with slight neuronal loss. In the medial shell, GFAP-immunopositive astrocytes were particularly distributed in the striosome portion, which is associated with a subset of limbic pathways. The VTA showed mild gliosis along the dopaminergic neurons, but without prominent neuronal loss in the region. In frontal cortex regions such as the OFC and ACC, neuronal deformation with cytoplasmic shrinkage and ischemic changes in neurons were seen, although the severity varied from case to case. VTA dopamine neurons project not only to the NAcc, but also to other limbic-related regions including the frontal cortex, hippocampus, and amygdala. 21 In both ill and recovered AN cases, previous imaging studies have supported the notion that abnormal anatomical connectivity exists between the NAcc and frontal cortex, based on resting-state images, 22 and, in addition, connectivity between the frontal cortex, insula, and NAcc has been observed in recovered AN cases. 9 Astrocytes have a range of functions in the central nervous system. 23 In particular, astrocytic protrusions surrounding synapses, called perisynaptic astrocytic processes, are involved in synapse maturation and stability, 24 and correlate with the pathophysiology of depression 25 and schizophrenia. 26 In this study, AN cases showed GFAP-positive astrocytic protrusions, as well as astrocytic cells, in the NAcc and related regions. Overall, our results might indicate that NAcc pathology induces retrograde axonal degeneration in the mesolimbic pathway in AN. It seems plausible that the NAcc or VTA pathology is associated with a deficit of the reward system, although we cannot rule out an effect of hypoxic encephalopathy before death.
In this context, the presence of GFAP fragments in the NAcc might be related to disease-specific abnormalities in AN. Interestingly, similar results have been reported in other neurodegenerative diseases. Human brain immunoblots from a case of traumatic brain injury revealed a cluster of bands from 38 to 50 kDa, which were identified as GFAP and GFAP breakdown products, and the presence of GFAP autoantibodies was suggested. 27 Another report described the detection of proteolyzed fragments of GFAP in Western blots of autopsied striatum and SN of patients with multiple-system atrophy and progressive supranuclear palsy, together with tau-positive neurofibrillary tangles, and the authors suggested that astrogliosis might protect specific regions from damage. 28 In animal studies of AN, some authors have reported reduced numbers of GFAP-immunoreactive astroglial cells in the cortexes, and it has been proposed that starvation-induced neuropsychological changes may occur in patients with AN. 29,30 However, all human postmortem studies of AN have found astrogliosis in the cortexes with neuronal degeneration. 13,31 We consider that these astroglial changes might correlate with the severity or duration of the illness, and might be associated with a deficit of the neuronal pathways. In other words, we suggest that these changes may be a pathophysiological feature of AN.
Consequences of prolonged severe physiological stress in AN Various mechanisms could lead to brain alterations in prolonged AN. One possibility is impaired cerebrovascular function associated with fluid shifts between the circulatory system and the interstitial space, resulting in an increased cerebrospinal fluid volume. 32 Low albumin levels have been associated with alterations in fluid shifts 33 and, indeed, most of our cases presented low albumin values. Notably, cases with idiopathic normal pressure hydrocephalus exhibit astrogliosis in the perivascular regions with enlarged ventricles, 34,35 and the pathophysiology is reversible, as in prolonged AN. It is considered that ventricular enlargement stretches and compresses the periventricular regions, leading to interstitial edema or progressive axonal loss. 36,37 Both disorders also exhibit demyelination of the frontal lobes and arteriolar sclerosis of the periventricular white matter, 13,31 although the underlying mechanisms have not been clarified. Furthermore, the Wernicke encephalopathy-like pathology of some of our cases, caused by vitamin B1 deficiency, has already been described in connection with AN. 31,38 The characteristic features of AN include gliosis, swelling of capillary endothelial cells, and capillary proliferation in the parenchymal regions. In addition, some cases showed central pontine myelinolysis, hypoxia caused by anemia, and hypoglycemia. 31,39 Multiple factors are involved in the pathophysiology of prolonged AN, and combined approaches based on the behavioral, functional, and pathological changes of the neuroanatomical system are likely to be significant for future therapy.
Correlations between psychiatric symptoms and pathology in AN It is well recognized that patients with AN who have a long disease duration show multiple psychiatric symptoms and are less likely to respond to psychological treatments. 5 The cases in our study also presented various psychiatric symptoms with relatively long duration, especially cognitive rigidity, which might reflect a deficit of the reward pathway. Studies of other neurodegenerative conditions indicate that pathological changes in the pathway-related regions might induce specific psychiatric symptoms as a result of a reward circuit deficit. 14,40 It is also possible that altered serotonin (5-hydroxytryptamine) function contributes to multiple psychiatric symptoms in AN, 5 and serotonin-dopamine interactions might be involved. Multiple psychiatric symptoms in AN may be associated with dysregulations of various neurotransmission pathways. It would be valuable to investigate whether the neuropathological findings observed in the present study are disease-specific or are consequences of chronic malnutrition.

Limitation
We could not obtain age-matched healthy female controls in this study, as the archives in our psychiatric or neurological hospitals did not contain suitable cases. Nevertheless, our AN cases showed significantly higher levels of GFAP-immunoreactive astrocytes than the aged control cases, although it should be borne in mind that periventricular astrogliosis sometimes occurs in aged persons.

Conclusion
The cases with prolonged AN showed massive astrogliosis in the shell compartment of the NAcc and VTA. These findings may indicate that impaired functional connectivity in the reward-related network induces neuropsychiatric symptoms in AN. Structural brain abnormalities have been considered to be reversible in individuals with eating disorders, but prolonged illness might induce irreversible brain pathology. Multiple factors are certainly involved in the pathophysiology of prolonged AN. However, we consider that our results regarding the dopaminergic pathway reflect one aspect of the pathophysiology in AN, even though we cannot rule out the possibility of cachexia caused by malnutrition. Relatively new imaging modalities, such as diffusion tensor imaging, may be useful to detect axonal loss and astrogliosis for the differential diagnosis of AN. Drugs targeting dopamine receptors or astrocytic functions might be candidates for the treatment of AN in the future. 41 AMED (JP18dm0908001 to K.O.), AMED (JP20dm0107108 to S.I.), and AMED (JP20dm0107105 to M.Y.