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Histopathological changes in the pituitary glands of female hamsters infected with the 139H strain of scrapie
J. Comp. Path. 1996 Vol. 114, 291-304
Histopathological Changes in the Pituitary Glands of Female Hamsters Infected with the 139H Strain of Scrapie X. Ye a n d R. I. Carp New York State Institutefor Basic Research in Developmental Disabilities, 1050 Forest Hill Roa6 Staten Island, N Y 10314, USA
Summary Previous studies in hamsters showed that the 139H strain of scrapie injected intracerebrally caused a generalized endocrinopathy and marked hypoglycaemia and hyperinsulinaemia. The low scrapie infectivity levels in the pancreas suggested that the changes noted in that organ were of neuroendocrine origin. In the current study, female weanling Syrian hamsters were inoculated intracerebrally with scrapie strain 139H or 263K, or with homogenate of normal hamster brain. Coronal sections of the pituitary gland were stained with haematoxyfin and eosin, Gomori's one-step trichrome, Congo red, thioflavin-S, and antibodies specific for several pituitary hormones. Sections were examined by light microscopy. The hamsters inoculated with scrapie strain 139H showed extensive pituitary vacuolization. Most vacuoles were located in the ventral or ventrolateral parts of the pars distalis. The pituitary glands of 139H-infected hamsters also showed cellular changes, namely, hypertrophy, atrophy and cytoplasmic vesicles. Nuclear changes such as swelling, vesicle formation, chromatin increase, pyknosis, karyorrhexis and karyolysis also occurred. The cellular and nuclear changes were most pronounced in the regions with vacuolation. Hamsters infected with the 263K strain did not show these changes. Immunocytochemical examination suggested that parenchymal cell types which produce different hormones were affected in areas of vacuolation. The changes produced by 139H were not seen in hamsters infected with strain 263K. This study provides the first evidence of cytopathological changes in the pituitary glands ofscrapie-infected animals and suggests a relationship between the pituitary changes and the pathological findings in the pancreas and other endocrine organs of 139Hinfected hamsters.
9 1996 W.B. Saunders Company Limited
Introduction Scrapie is a neurodegenerative disease occurring naturally in sheep and goats (Dickinson, 1977). It has an insidious onset, beginning with the animal scraping its fleece against fixed objects. The disease progresses to tremors and ataxia. It culminates in death within 6 weeks to 6 months. The scrapie agent has been passaged in a number of laboratory animal species, including mice and hamsters (Chandler, 1963; Kimberlin and Walker, Correspondence to: X. Ye, Division ofNeurotoxicologT, FDA/National Center for Toxicological Research, 3900 NCTR Drive, Jefferson, AR 72079, USA. 0021-9975/96/030291 + i4 $12.00/0
9 1996 W.B. Saunders Company Limited
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x. Ye and R. I. Caxp
1986). The biological characteristics of a series ofscrapie strains were described by Carp et al. (1994). Pathological changes in the brains of infected animals depend upon interaction between the scrapie strain and host. Carp et al. (1990) showed that in some scrapie strain-mouse strain or scrapie strain-hamster strain combinations, there was an increase in body weight; this began before the onset of motor dysfunction, which typically signals the start of clinical disease. For this early study, hamsters were inoculated intracerebrally with scrapie strains 139H or 22CH, or with homogenate of normal hamster brain. During the latter part of the pre-clinical and whole of the clinical phase of disease, animals were hypoglycaemic and showed marked hyperinsulinaemia, with insulin values as much as 49-fold higher than those seen in controls. At necropsy, there was marked hyperplasia and hypertrophy of the cells of the islets of Langerhans, and the thyroid glands, adrenal glands, liver and kidneys were increased in size and weight. In contrast, hamsters inoculated with the commonly used 263K strain of hamster-adapted scrapie did not show any of the changes described above. The differences observed between 139H- or 22CH- and 263K-infected hamsters may have been due to variation between strains in terms of the brain regions or neuronal cell types that they affect (Carp et al., 1990, 1994). It is our hypothesis that 139H-induced obesity and hyperinsulinaemia is mediated by scrapie-induced damage to the control centres of the central nervous system, including the hypothalamus and pituitary gland. In the present study, histochemical and immunolabelling methods were used to evaluate histopathological changes in the pituitary glands of scrapieinfected hamsters. PrP so, the major protein associated with scrapie infectivity and neuropathogenesis, is derived from the normal cellular isoform, PrP c, during scrapie infection (Prusiner and DeArmond, 1994). Accordingly, we tested the scrapie infectivity of the pituitary gland and used Congo red, thioflavin-S and PrP sc immunocytochemical staining to assess PrP sc amyloid formation. Materials and Methods Animals
Female weanling LVG/LAK hamsters (Charles River, Wilmington, MA, USA) were maintained in a temperature- and humidity-controlled room with a 12-h on/12-h off light cycle. Each cage contained no more than three animals. The hamsters were given food and water ad libitum. Inocula
Three inocula were used: homogenates of normal hamster brain (NHB), scrapie strain 139H and scrapie strain 263K. The scrapie strains were kindly provided by R. H. Kimberlin (Scrapie and Related Diseases Advisory Service, Edinburgh, UK) and are now maintained by LVG/LAK hamster-to-hamster passage with 1% brain homogenates, which give mean incubation periods of 134 and 65 days with strains 139H and 263K, respectively. The characteristics of the passaged materials were the same as those of the strains obtained from Dr Kimberlin (Carp et al., 1990; Kascsak et al., 1991). Passages were made by the intracerebral injection of 40 gl of brain homogenate prepared in cold (4~ phosphate buffered saline (PBS), pH 7-4, by 20
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strokes of a hand-operated Ten-Broeck homogenizer (Carp et al., 1990). All homogenates were stored at --70~ until used. The animals were inoculated when c. 35 days old, 1-2 weeks after arrival at the laboratory.
Assay of Scrapie Infectivity Brain and pituitary gland of 139H- and 263K-inoculated hamsters were obtained towards the end of the clinical phase. Pituitary homogenates were diluted' to 1% and 0"01% concentrations (w/v) before being injected intracerebrally; brain homogenates were injected as 0" 1% and 0"001% homogenates. The occurrence of clinical disease and the incubation period were assessed as described by Carp et al. (1990).
7~ssue Preparation Paraffin-wax sections were used for histological analysis and for immunolabelling. At the end of the incubation period, animals were anaesthetized with pentobarbitone sodium (60 mg/ml; Abbott Laboratories, North Chicago, IL, USA), injected intraperitoneally at a dose rate of 3-4 ml/kg body weight. Anaesthetized animals were perfused through the heart with saline (0"9%) at room temperature for 2-3 min (15 ml/min) followed by paraformaldehyde 4% and glutaraldehyde 0-05% (Electron Microscopy Sciences, Ft Washington, PA, USA) in 0" 1M PBS, pH 7'4, for 10 15 min (15 ml/min). Animals were then decapitated. Brain and pituitary gland were removed, fixed by immersion in the perfusion solution for 24 h, and then washed in PBS for 2 3 days at 4~ Small blocks of tissue were put into labelled carriers. After washing, brain and pituitary blocks were dehydrated in graded alcohols and embedded in paraffin wax. Serial coronal (or sometimes sagittal) sections were mounted on gelatincoated slides and allowed to dry overnight at 37~ Adjacent sections were mounted on sequential slides.
Histological and Cytological Staining Methods Pituitary and brain sections were stained with haematoxylin and eosin (HE), Gomori's one-step trichrome, Congo red, and thioflavin-S. Gomori's one-step trichrome stains collagen fibres blue-green. Congo red stains amyloid orange and results in an intense green-red birefringence when viewed with polarized light. Amyloid stained with thioflavin-S fluoresces when observed with ultraviolet light. The slides were also observed by light microscopy.
Immunocytochemical Labelling This was performed as described by Ye et al. (1994b). Pituitary sections were stained with peroxidase anti-peroxidase (PAP) or alkaline phosphatase kits (BioGenex, San Ramon, CA, USA). In brief, before immunocytochemical staining, sections were dewaxed in xylene, placed in absolute ethanol, and then in H202 1% in methanol for 15 min to block endogenous peroxidase activity. The sections were rehydrated through graded ethanol solutions, rinsed in PBS, incubated for 30 rain with normal sheep serum (Cappel Organon Teknika Co., Durham, NC, USA) diluted 1 in 50 in PBS, and then incubated overnight at 4~ with primary antibody. Two antisera against adrenocorticotropic hormone (ACTH) were used: anti-ACTH1 14 (ICN Biomedical Inc., Lisle, IL, USA) and anti-ACTH17_39 (Peninsula Laboratories, Belmont, CA, USA). Each of these antisera stains "full length" ACTH; use of both provided confirmation of the specificity of the A C T H immunostaining. Antibodies to folliclestimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were obtained from BioGenex. Antibodies to thyroid-stimulating hormone (TSH) and growth hormone (GH) were from Chemicon International, Temecula, CA, USA. Rabbit polyclonal antiserum reacting with both hamster and mouse PrP was obtained
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from Dr R.J. Kascsak of this Institute. For PrP sc immunolabelling, sections were first treated for 15 rain with 88% formic acid (Diedrich et al., 1991). The antisera used in this study had already been tested for specificity. In the PAP method, after reaction with the primary antibody and rinsing, the sections were incubated with a secondary antibody (sheep, anti-rabbit serum; Cappel Organon Teknika Co., Durham, NC, USA) diluted 1 in 100 with PBS and incubated for 1 h at room temperature. After thorough rinsing with PBS, the sections were incubated for 1 h at room temperature with rabbit PAP (Cappel Organon Teknica Co.) diluted 1 in 100 with PBS, and then with diaminobenzidine substrate for 3 rain at room temperature. The slides were rinsed in distilled water, dehydrated in an ascending ethanol series, and mounted. With the alkaline phosphatase kits, after reaction with the primary antibody and rinsing, the sections were incubated sequentially at room temperature with biotinylated secondary antibody, alkaline phosphatase-conjugated streptavidin, and chromogen substrate (BioGenex). The slides were rinsed in distilled water and mounted in glycergel. Two controls for antibody specificity were used. (1) Negative control: specimens which did not contain the antigen to be stained were processed in the same way as the unknown. (2) Reagent control: pituitary sections were allowed to react with a non-immune serum (1 in 500) instead of a primary antibody. The control tests did not show any cellular staining.
Assessment of Immunolabelling A Quantimet 970 image analyser (Cambridge Instruments) was used to quantify the distribution and intensity of pituitary immunostaining. A stained slide was scanned by a scanner mounted on a microscope. To improve sensitivity, the Quantimet 970 includes an "interactive user-programming interface" (QUIPS/MX, Version V07.00). Data were analysed with the Lotus 1 2-3 program and the CSS statistics software packages. We obtained information on the size of the stained areas of the pars distalis (PD) in 139H-infected hamsters and on the optical density of these areas. This information was stored, analysed statistically and compared by Student's t-test with the data from sections obtained from animals inoculated with NHB homogenates. Results
Histopathology The pituitary gland consists of three parts, namely the pars nervosa (PN), pars intermedia (PI) and pars distalis (PD) (Figs 1 and 2). In control hamsters, the cells in the PD were closely packed together, forming thick branching cords of cells with numerous sinusoids (Si) located between them; occasionally, endothelial cells could be observed at the margins of the sinusoids. In 139Hinfected hamsters, there was extensive extracellular vacuolation in the pituitary glands. Most vacuoles were located in the ventral or ventrolateral parts of the PD. The pituitary cells were dispersed and the sinusoids damaged, especially in the area of vacuolation. In contrast, there was comparatively little vacuolation in the pituitary glands of 263K-infected hamsters (Figs 2b and 2d). As shown in Figs 1 and 2, the pituitary glands of 139H-infected hamsters also showed cellular hypertrophy, cellular atrophy and necrosis, and cytoplasmic vesicles. Nuclear changes included swelling, the appearance of ring-forms, vesicle formation, pyknosis, karyorrhexis and karyolysis. The cellular and nuclear pathological changes were most pronounced in regions with vacuolation. In contrast, there were comparatively few cellular changes in the pituitary glands of 263K-infected hamsters (Figs 2b and 2d). Use of Gomori's
Pituitary Changes in Scrapie .e
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"
--
i ~
Fig. 1.
~
-
.~
a-d. Haematoxylin and eosin stain of pituitary glands, a, b, Control hamster; c, d, 139H-infected hamster, a, x 65, b d, x 262. (PN, pars nervosa; PI, pars intermedia; PD, pars distalis). In the control hamster, the pituitary cells in the PD are closely packed with m a n y sinusoids (Si) located between them. However, in the 139H-infected hamster, the pituitary cells in the PD are more dispersed and many Si are destroyed. There are numerous vacuoles (*) located on the ventral or ventrolateral parts of the PD. Pituitary glands of 139H-infected hamsters show cellular hypertrophy and necrosis (arrowhead). Nuclear pathological changes are present, such as swelling (small arrow) and ring-form changes (medium arrow); vesicular changes, pyknosis, karyorrhexis and kar,volysis (arrowhead) are also evident. The cellular and nuclear pathological changes are most pronounced in the regions with vacuolation.
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Fig. 2.
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~ d . Pituitary glands in 139H- and 263K-infected hamsters stained with Gomori's one-step trichrome stain; a, c, 139H-infected hamster; b, d, 263K-infected hamster, a, b, x 321 c, d, x 262. (PI, pars intermedia; PN, pars nervosa; PD, pars distalis). There is extensive extracellular vacuolation (*) and cellular atrophy (arrowhead) in the ventral and ventrolateral parts of the PD in the 139H-infected hamster, with comparatively little vacuolation in the 263K-infected hamster. No abnormal collagen fibres are visible in the pituitary glands of these scrapie-infected hamsters.
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one-step trichrome failed to show any abnormal collagen fibres in the pituitary glands of 139H- or 263K-infected hamsters. Both Congo red and thioflavinS stains failed to show evidence of amyloid formation.
Immunocytochemistry To unmask epitopes of the aggregated abnormal isoform of the scrapie protein, sections were treated with 88% formic acid for 15 min before being allowed to react with the primary antiserum for PrP so. With this technique we previously demonstrated PrP sc formation and amyloid plaques in the cortex, hippocampus and substantia nigra, and around the pia mater, corpus callosum, fimbria, ventricles and blood vessels in sections from 263K-infected animals (Ye et al., 1993). In the present study, PrP sc immunolabelling was not seen in the pituitary glands of scrapie-infected or control animals. To investigate the cell types affected by scrapie infection, the immunolabelling patterns of several hormones were examined in the pituitary glands of control and 139H- and 263K-infected hamsters (Figs 3-5). In control animals, cells giving a positive reaction for ACTHI_24, ACTHI7 39, GH, T S H and PRL were distributed throughout the PD. The intensity of the immunostaining varied, even in sections stained with the same primary antibody. Some of the positive cells were located around the sinusoids. As shown in Figs 3 and 5, the immunostaining patterns of ACTHl_24 did not differ dramatically in 139H-infected, 263K-infected and control animals, positively reacting cells being found in the PI and PD but not in the PN. However, there were fewer ACTH1 24-immunostained cells in the extracellular vacuolation areas of the PD of 139H-infected hamsters than of other animals (Figs 3b and 3d). Control and 139H-infected hamsters differed slightly in respect of the pituitary immunostaining patterns of T S H (Figs 4 and 5) and PRL (not shown). TSH-immunostained cells were found primarily in the PD, with a few positive cells in the PI and none in the PN of either control or scrapie-infected hamsters. PRL-immunostained cells were found only in the PD. The TSHand PRL-immunostained cells were fewer in the PD vacuolation areas of 139H-infected hamsters than in control animals. As shown in Figs 4 and 5, the GH immunostaining patterns were similar in all three groups of hamsters. GH-positive cells were found mainly in the PD, but a few were seen in the PI, and immunostained fibres were seen in the PN. GH-immunostained cells occurred less frequently in the ventrolateral area than in other parts of the PD; since this was the area that showed the most extensive histopathological changes, it was not surprising that the effect on G H immunostaining in 139H-infected animals was minimal (Fig. 4 and Table 1). The immunostaining patterns of ACTHl_24, ACTH17 ~9, G H and T S H in the pituitary glands of 263K-infected hamsters are shown in Fig. 5. Vacuolation was not present and the patterns of these hormones and PRL were similar to those seen in the controls. There were only a few FSH- and LH-immunostained cells in the pituitary
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Fig. 3.
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~ d . Immunostaining patterns of ACTttj_24 in the PD of pituitary glands, a, Control hamster; b, c, d, 139H-infected hamster, a, b, x 65, c, x 131, d, x 262. The immunostaining ofACTH~ 24 is distributed throughout the pars distalis (PD) and pars intermedia (PI) in both the control hamster and the 139H-infected hamster. Arrowhead shows intensive ACTHI 24 immunostaining (ir-ACTHI 24) in PD and weak staining in PI. The number of cells stained for ACTHI-24 was reduced in those focal areas (*) that had extensive vacuolation in the 139H-infected pituitary gland.
Pituitary
Fig. 4.
Changes
in Scrapie
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~ d . Immunostaining patterns of T S H and GH in the PD of pituitary glands, a, x 65, TSHimnmnostaining (ir-TSH), control hamster; b, x 131, ir-TSH, 139FI-infected hamster; c, x 65, GH-immunostaining (ir-GH), control hamster; d, x 131, ir-GH, 139H-infected hamster; (PI, pars intermedia; PD, pars distalis; *, vacuolation area). The immunostaining of T S H and GH is distributed throughout the PD in the control hamster and the 139H-infected hamster. Arrowheads show T S H and G H immunostaining in PD and PI. GH-immunolabelled cells are more rare in the ventral and ventrolateral area than in other parts of the PD in both control and 139H-infected hamsters.
300
Fig. 5.
X . Ye a n d R . I. C a r p
a d. Immunostaining patterns of ACTHl-24, ACTHI7 39, G H and T S H in the pituitary glands of 263K-infected hamsters, a, ACTHI 24; b, ACTI-II7_39; C, GH; d, TSH; ~ d , x 65. (PI, pars intermedia; PD, pars distalis). The pituitary glands of 263K-infected hamsters do not show vacuolation in the PD as is seen in 139H-infected hamsters. Arrowheads show immunostaining in PD and PI. The immunostaining patterns with different antisera are very similar in the pituitary glands of the control and 263K-infected hamsters (Fig. 3a, Fig. 4a, c). The immunostaining patterns of A C T H I 24 and ACTHI7 ~9 are very similar to each other in both PD and PI, which suggests that both of the antisera stained cells containing full length A C T H .
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Pituitary Ghanges in Scrapie Table 1 I m m u n o s t a i n i n g of the pituitary glands o f 139H-infected and control h a m s t e r s Inoculum
Number of hamsters
NHB 139H NHB 139H NHB 139H NHB 139H NHB 139H NHB 139H NHB 139H
Immunostain target (hormone*)
10 11 11 14 15 13 15 18 11 15 12 15 13 14
Immunostaining results (+_ SEM) OD
ACTH~7 39 " ACTH~ 24 " GH " TSH " PRL " FSH " LH "
0"30 0"30 0'33 0'34 0'27 0"30 0'25 0"27 0"20 0'19 0'13 0'17 0'03 0"04
PA (mm 2)
(0"02) (0"01) (0"02) (0"02) (0"02) (0"02) (0"03) (0"02) (0"02) (0'02) (0'04) (0"02) (0'02) (0'02)
0"28 0'20 0"27 0"23 0'26 0"36 0"20 0"22 0'23 0"32 0'02 0"02 0"00 0"00
PA %
(0"03) (0"04) (0"03) (0"03) (0"04) (0"04) (0"02) (0"02) (0"06) (0"06) (0"01) (0"01) (0"00) (0"00)
11"9 9-6 13"1 11"6 11'1 13"0 9"0 9"4 10'9 14"2 0"9 1"2 0"l 0"0
(1"0) (1"6) (1'3) (1"3) (1"6) (1"7) (1"1) (0"8) (3'6) (2"6) (0"4) (0"5) (0"1) (0"0)
*Abbreviations defined in Materials and Methods. NHB, normal hamster brain; OD, optical density; PA, positively stained area of pars distalis; PA%, positively stained area expressed as a percentage of the whole pars distalis.
Table 2 Histopathological changes, infectivity titres and PrP concentrations in the pituitary gland and pancreas of 263K- and 139H-infected h a m s t e r s Organ
Pituitary gland Pancreas~
Histopathological changes
Infectivity titre* (mean)
PrP concentration
263K
139H
263K
139H
262K
139H
+
+ + +
6"3
8"8
ND
ND
-
+ + +
>3"9<5"4
>3'9<5"4
Low
High
* Log~0. "~Results from the studies of Carp et al. (1990, 1994) and Ye et al. (1994a). ND, not done; + + + , striking; _+, slight; - , absent.
glands of scrapie-infected and control animals. Most of the LH-positive cells were located at the edge of the ventral PD (not shown). Pituitary immunostaining results obtained from 139H-infected and control hamsters were compared with regard to optical density and to the positively stained area expressed as a percentage of the whole PD (Table 1). The results showed no significant differences (unpaired t-test; P>0"05). However, as noted above, there was a reduced number o f A C T H - , TSH- and PRL-immunostained cells in the focal areas of vacuolation seen in 139H-infected hamsters. Table 2 summarizes the data now available on the effects of 263K and 139H on the pituitary gland and pancreas. In the pituitary gland the 139H strain differed from the 263K strain in producing more severe histopathological changes and higher infectivity titres.
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Discussion The pituitary gland is structurally and functionally complex, controlling many other endocrine glands in the body. The synthesis and secretion of many pituitary hormones, such as PRL, FSH, LH, A C T H and GH, are influenced by a variety of stimuli (e.g., sleep, stress) and are episodic and pulsatile. Plasma concentrations of such hormones can change dramatically within minutes. This is especially true in females, because of the oestrous cycle. This may explain the high degree of variation in immunostaining patterns seen within single groups of the female hamsters used in these studies (Table 1). It seems possible that the obesity and hyperinsulinaemia induced in hamsters by the 139H strain of scrapie may be mediated by organs such as the hypothalamus or pituitary. In a previous study, 139H-infected hamsters showed a significant increase in neurons staining for a corticotropin-releasing factor (CRF) in the preoptic nucleus and a Significant decrease in neurons staining for vasopressin (VP) in the lateral hypothalamus (Ye et al., 1994c). The hypothalamus is an important control organ for the neuroendocrine system. Hypothalamic hormones are released from hypothalamic nerve fibre endings around capillaries in the pituitary stalk and reach the anterior lobe (pars distalis) through the special portal system. In addition, hypothalamic-releasing factors can stimulate the synthesis and release of pituitary tropic hormones, which can regulate the activities and functions of other endocrine organs. Both CRF and VP can stimulate the release of A C T H from the pituitary gland. Abnormal CRF and VP activity is capable of disturbing pituitary cellular functions and morphology. Abnormal changes in the islets of Langerhans, such as hypertrophy and hyperplasia of B cells, may be directly responsible for the obesity and hyperinsulinaemia reported in 139H-infected hamsters (Ye eta[., 1994a, b). The cause of lesions of the islets of Langerhans may be "local" (originating within the cells of the islets) or "central" (changes in the brain). Scrapie strain 139H can induce different pathological patterns in different organs of the hamster (Carp et al., 1993, 1994). Since scrapie infectivity titres in these organs (e.g., pancreas and adrenals) are low, it is unlikely that the scrapie agent directly causes pathological changes in organs outside the central nervous system. Such changes are probably induced indirectly and are a function of the specific "targeting" effect of the 139H strain in the central nervous system (Carp et al., 1990). If the lesions of the islets of Langerhans are based "centrally" on lesions in the brain, two possible pathways should be considered: hormonal and neuronal. In the neuronal pathway, lesions in the hypothalamus would stimulate the motor neurons of the vagus in the brain stem, increasing parasympathetic activity. Acetylcholine released from the vagus nerve can stimulate B-cell activity, and insulin-secretion-promoting factor released from the ventral hypothalamus can stimulate insulin release (Jeanrenaud, 1985; Bobbioni-Harsch and Jeanrenaud, 1989). An anterior pituitary hormone, galanin, can inhibit insulin secretion (Kaplan et al., 1988). In 139H-infected hamsters these hormones may be affected by the cellular damage in the hypothalamus and pituitary gland.
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Hamsters infected with the 139H scrapie strain showed extensive vacuolation in the pituitary glands, while the glands in 263K-infected hamsters showed little or none. There is a significant difference in incubation period between the two scrapie strains (139H, 135 days; 263K, 65 days). We do not know whether this difference could account for the more pronounced pituitary lesions in the 139H-infected hamsters. The different phenotypes seen in hamsters infected with one or other of the two scrapie strains (Carp et al., 1994), and the pathological changes seen in the pituitary glands of 139Hinfected but not 263K-infected hamsters, suggest that these two scrapie strains have different effects on either brains cells or pituitary cells. In the pituitary gland, strain 139H, unlike 263K, produced striking histopathological changes and high infectivity titres (Table 2), but neither strain gave rise to demonstrable degrees of amyloid formation or PrP sc accumulation. The degree of PrP sc accumulation in the pituitary glands was clearly less than that seen in the brain of scrapie-infected hamsters; infectivity titres were also lower in the pituitary gland than those that occur in the brain, particularly in 263Kinfected animals. In conclusion, this study supports a possible role of a hypothalamic or a pituitary factor in mediating the obesity and hyperinsulinaemia produced by infection with the scrapie 139H strain.
Acknowledgments We thank Drs A. Scallet, J. Fikes, and B. Bolon for critical reading of this manuscript. X. Ye was supported in part by the New York State Department of Mental Hygiene, in part by NIH grant AG0917, and in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. References Bobbioni-Harsch, E. andJeanrenaud, B. (1989). The hypothalamic origin of an insulin secretion promoting factor present in the plasma of normal rats. Journal of Neuroendocrinology, 1, 103 108. Carp, R. I., Kascsak, R.J. and Rubenstein, R. (1993). Pathogenesis of unconventional slow infections. In: Light and Electron Microscopic Neuropathology of Slow Firus Disorders, P. P. Liberski, Ed., CRC Press Inc., Boca Raton, Florida, pp. 33 61. Carp, R. I., Kim, Y. S. and Callahan, S. M. (1990). Pancreatic lesions and hypoglycemia-hyperinsulinemia in scrapie-injected hamsters. Journal of Infectious Diseases, 161,462 466. Carp, R. I., Ye, X., Kascsak, R. J. and Rubenstein, R. (1994). The nature of the scrapie agent: biological characteristics of scrapie in different serapie strain host combinations. In: Slow Infections of the Central Nervous System, J. Bjornsson, R. I. Carp, A. L6ve, and H. M. Wisniewski, Eds, Annals of the New York Academy of Sciences, 724, 221 234. Chandler, R. L. (1963). Experimental scrapie in the mouse. Research in Veterinary Science, 4, 276-285. Dickinson, A. G. (1977). Scrapie in sheep and goats. In: Slow Virus Diseases of Animals and Man, R. H. Kimberlin, Ed., North-Holland, Amsterdam, pp. 209 241. Diedrich, J. F., Bendheim, P. E., Kim, Y. S., Carp, R. I. and Haase, A. T.
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(1991). Scrapie associated prion protein accumulates in astrocytes during scrapie infection. Proceedingsof the National Academy of Sciences USA, 88, 375 379. Jeanrenaud, B. (1985). An hypothesis on the aetiology of obesity: dysfunction of the central nervous system as a primary cause. Diabetologia, 28, 502-513. Kaplan, L. M., Gabriel, S. M., Koenig, J. I., Sunday, M. E., Spindel, E. R., Martin, J. B. and Chin, W. W. (1988). Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proceedingsof the National Academy of Sciences USA, 85, 7408-7412. Kascsak, R. J., Rubenstein, R. and Carp, R. I. (1991). Evidence for biological and structural diversity among scrapie strains. Current Topics in Microbiology and Immunology, 172, 139-152. Kimberlin, R. H. and Walker, C. A. (1986). Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly. Journal of General Firology,67, 255-263. Prusiner, S. B. and DeArmond, S.J. (1994). Prion diseases and neurodegeneration. Annual Researchof Neuroscience, 17, 311-339. Ye, X., Carp, R. I., Kascsak, R., Kozielski, R. and Kozlowski, P. (1993). Astrocytosis and amyloid deposition in scrapie-infected hamsters. In: Biology and Pathology of Astrocyte-Neuron Interactions, S. Fedoroff, B. H. J. Juurlink and R. Doucette, Eds, Plenum Press, New York and London, p. 443 (Abstract). Ye, X., Carp, R. I., Yu, Y., Kozielski, R. and Kozlowski, P. (1994a). Histopathological changes in the islets of Langerhans in 139H-infected hamsters. Journal of Comparative Pathology, 110, 153-167. Ye, X., Carp, R. I., Yu, Y., Kozielski, R. and Kozlowski, P. (1994b). Hyperplasia and hypertrophy of B-cells in the islets of Langerhans in hamsters infected with the 139H strain of scrapie. Journal of ComparativePathology, 110, 169 183. Ye, X., Carp, R. I., Yu, Y., Kozielski, R. and Kozlowski, P. (1994c). Effect of infection with the 139H scrapie strain on the number, area and/or location ofhypothalamic CRF- and VP-immunostained neurons. Acta Neuropathologica, 88, 44-54.
Received, September8th, 1995] Accepted, December4th, 1995 J
Histopathological Changes in the Pituitary Glands of Female Hamsters Infected with the 139H Strain of Scrapie X. Ye a n d R. I. Carp New York State Institutefor Basic Research in Developmental Disabilities, 1050 Forest Hill Roa6 Staten Island, N Y 10314, USA
Summary Previous studies in hamsters showed that the 139H strain of scrapie injected intracerebrally caused a generalized endocrinopathy and marked hypoglycaemia and hyperinsulinaemia. The low scrapie infectivity levels in the pancreas suggested that the changes noted in that organ were of neuroendocrine origin. In the current study, female weanling Syrian hamsters were inoculated intracerebrally with scrapie strain 139H or 263K, or with homogenate of normal hamster brain. Coronal sections of the pituitary gland were stained with haematoxyfin and eosin, Gomori's one-step trichrome, Congo red, thioflavin-S, and antibodies specific for several pituitary hormones. Sections were examined by light microscopy. The hamsters inoculated with scrapie strain 139H showed extensive pituitary vacuolization. Most vacuoles were located in the ventral or ventrolateral parts of the pars distalis. The pituitary glands of 139H-infected hamsters also showed cellular changes, namely, hypertrophy, atrophy and cytoplasmic vesicles. Nuclear changes such as swelling, vesicle formation, chromatin increase, pyknosis, karyorrhexis and karyolysis also occurred. The cellular and nuclear changes were most pronounced in the regions with vacuolation. Hamsters infected with the 263K strain did not show these changes. Immunocytochemical examination suggested that parenchymal cell types which produce different hormones were affected in areas of vacuolation. The changes produced by 139H were not seen in hamsters infected with strain 263K. This study provides the first evidence of cytopathological changes in the pituitary glands ofscrapie-infected animals and suggests a relationship between the pituitary changes and the pathological findings in the pancreas and other endocrine organs of 139Hinfected hamsters.
9 1996 W.B. Saunders Company Limited
Introduction Scrapie is a neurodegenerative disease occurring naturally in sheep and goats (Dickinson, 1977). It has an insidious onset, beginning with the animal scraping its fleece against fixed objects. The disease progresses to tremors and ataxia. It culminates in death within 6 weeks to 6 months. The scrapie agent has been passaged in a number of laboratory animal species, including mice and hamsters (Chandler, 1963; Kimberlin and Walker, Correspondence to: X. Ye, Division ofNeurotoxicologT, FDA/National Center for Toxicological Research, 3900 NCTR Drive, Jefferson, AR 72079, USA. 0021-9975/96/030291 + i4 $12.00/0
9 1996 W.B. Saunders Company Limited
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x. Ye and R. I. Caxp
1986). The biological characteristics of a series ofscrapie strains were described by Carp et al. (1994). Pathological changes in the brains of infected animals depend upon interaction between the scrapie strain and host. Carp et al. (1990) showed that in some scrapie strain-mouse strain or scrapie strain-hamster strain combinations, there was an increase in body weight; this began before the onset of motor dysfunction, which typically signals the start of clinical disease. For this early study, hamsters were inoculated intracerebrally with scrapie strains 139H or 22CH, or with homogenate of normal hamster brain. During the latter part of the pre-clinical and whole of the clinical phase of disease, animals were hypoglycaemic and showed marked hyperinsulinaemia, with insulin values as much as 49-fold higher than those seen in controls. At necropsy, there was marked hyperplasia and hypertrophy of the cells of the islets of Langerhans, and the thyroid glands, adrenal glands, liver and kidneys were increased in size and weight. In contrast, hamsters inoculated with the commonly used 263K strain of hamster-adapted scrapie did not show any of the changes described above. The differences observed between 139H- or 22CH- and 263K-infected hamsters may have been due to variation between strains in terms of the brain regions or neuronal cell types that they affect (Carp et al., 1990, 1994). It is our hypothesis that 139H-induced obesity and hyperinsulinaemia is mediated by scrapie-induced damage to the control centres of the central nervous system, including the hypothalamus and pituitary gland. In the present study, histochemical and immunolabelling methods were used to evaluate histopathological changes in the pituitary glands of scrapieinfected hamsters. PrP so, the major protein associated with scrapie infectivity and neuropathogenesis, is derived from the normal cellular isoform, PrP c, during scrapie infection (Prusiner and DeArmond, 1994). Accordingly, we tested the scrapie infectivity of the pituitary gland and used Congo red, thioflavin-S and PrP sc immunocytochemical staining to assess PrP sc amyloid formation. Materials and Methods Animals
Female weanling LVG/LAK hamsters (Charles River, Wilmington, MA, USA) were maintained in a temperature- and humidity-controlled room with a 12-h on/12-h off light cycle. Each cage contained no more than three animals. The hamsters were given food and water ad libitum. Inocula
Three inocula were used: homogenates of normal hamster brain (NHB), scrapie strain 139H and scrapie strain 263K. The scrapie strains were kindly provided by R. H. Kimberlin (Scrapie and Related Diseases Advisory Service, Edinburgh, UK) and are now maintained by LVG/LAK hamster-to-hamster passage with 1% brain homogenates, which give mean incubation periods of 134 and 65 days with strains 139H and 263K, respectively. The characteristics of the passaged materials were the same as those of the strains obtained from Dr Kimberlin (Carp et al., 1990; Kascsak et al., 1991). Passages were made by the intracerebral injection of 40 gl of brain homogenate prepared in cold (4~ phosphate buffered saline (PBS), pH 7-4, by 20
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strokes of a hand-operated Ten-Broeck homogenizer (Carp et al., 1990). All homogenates were stored at --70~ until used. The animals were inoculated when c. 35 days old, 1-2 weeks after arrival at the laboratory.
Assay of Scrapie Infectivity Brain and pituitary gland of 139H- and 263K-inoculated hamsters were obtained towards the end of the clinical phase. Pituitary homogenates were diluted' to 1% and 0"01% concentrations (w/v) before being injected intracerebrally; brain homogenates were injected as 0" 1% and 0"001% homogenates. The occurrence of clinical disease and the incubation period were assessed as described by Carp et al. (1990).
7~ssue Preparation Paraffin-wax sections were used for histological analysis and for immunolabelling. At the end of the incubation period, animals were anaesthetized with pentobarbitone sodium (60 mg/ml; Abbott Laboratories, North Chicago, IL, USA), injected intraperitoneally at a dose rate of 3-4 ml/kg body weight. Anaesthetized animals were perfused through the heart with saline (0"9%) at room temperature for 2-3 min (15 ml/min) followed by paraformaldehyde 4% and glutaraldehyde 0-05% (Electron Microscopy Sciences, Ft Washington, PA, USA) in 0" 1M PBS, pH 7'4, for 10 15 min (15 ml/min). Animals were then decapitated. Brain and pituitary gland were removed, fixed by immersion in the perfusion solution for 24 h, and then washed in PBS for 2 3 days at 4~ Small blocks of tissue were put into labelled carriers. After washing, brain and pituitary blocks were dehydrated in graded alcohols and embedded in paraffin wax. Serial coronal (or sometimes sagittal) sections were mounted on gelatincoated slides and allowed to dry overnight at 37~ Adjacent sections were mounted on sequential slides.
Histological and Cytological Staining Methods Pituitary and brain sections were stained with haematoxylin and eosin (HE), Gomori's one-step trichrome, Congo red, and thioflavin-S. Gomori's one-step trichrome stains collagen fibres blue-green. Congo red stains amyloid orange and results in an intense green-red birefringence when viewed with polarized light. Amyloid stained with thioflavin-S fluoresces when observed with ultraviolet light. The slides were also observed by light microscopy.
Immunocytochemical Labelling This was performed as described by Ye et al. (1994b). Pituitary sections were stained with peroxidase anti-peroxidase (PAP) or alkaline phosphatase kits (BioGenex, San Ramon, CA, USA). In brief, before immunocytochemical staining, sections were dewaxed in xylene, placed in absolute ethanol, and then in H202 1% in methanol for 15 min to block endogenous peroxidase activity. The sections were rehydrated through graded ethanol solutions, rinsed in PBS, incubated for 30 rain with normal sheep serum (Cappel Organon Teknika Co., Durham, NC, USA) diluted 1 in 50 in PBS, and then incubated overnight at 4~ with primary antibody. Two antisera against adrenocorticotropic hormone (ACTH) were used: anti-ACTH1 14 (ICN Biomedical Inc., Lisle, IL, USA) and anti-ACTH17_39 (Peninsula Laboratories, Belmont, CA, USA). Each of these antisera stains "full length" ACTH; use of both provided confirmation of the specificity of the A C T H immunostaining. Antibodies to folliclestimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were obtained from BioGenex. Antibodies to thyroid-stimulating hormone (TSH) and growth hormone (GH) were from Chemicon International, Temecula, CA, USA. Rabbit polyclonal antiserum reacting with both hamster and mouse PrP was obtained
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from Dr R.J. Kascsak of this Institute. For PrP sc immunolabelling, sections were first treated for 15 rain with 88% formic acid (Diedrich et al., 1991). The antisera used in this study had already been tested for specificity. In the PAP method, after reaction with the primary antibody and rinsing, the sections were incubated with a secondary antibody (sheep, anti-rabbit serum; Cappel Organon Teknika Co., Durham, NC, USA) diluted 1 in 100 with PBS and incubated for 1 h at room temperature. After thorough rinsing with PBS, the sections were incubated for 1 h at room temperature with rabbit PAP (Cappel Organon Teknica Co.) diluted 1 in 100 with PBS, and then with diaminobenzidine substrate for 3 rain at room temperature. The slides were rinsed in distilled water, dehydrated in an ascending ethanol series, and mounted. With the alkaline phosphatase kits, after reaction with the primary antibody and rinsing, the sections were incubated sequentially at room temperature with biotinylated secondary antibody, alkaline phosphatase-conjugated streptavidin, and chromogen substrate (BioGenex). The slides were rinsed in distilled water and mounted in glycergel. Two controls for antibody specificity were used. (1) Negative control: specimens which did not contain the antigen to be stained were processed in the same way as the unknown. (2) Reagent control: pituitary sections were allowed to react with a non-immune serum (1 in 500) instead of a primary antibody. The control tests did not show any cellular staining.
Assessment of Immunolabelling A Quantimet 970 image analyser (Cambridge Instruments) was used to quantify the distribution and intensity of pituitary immunostaining. A stained slide was scanned by a scanner mounted on a microscope. To improve sensitivity, the Quantimet 970 includes an "interactive user-programming interface" (QUIPS/MX, Version V07.00). Data were analysed with the Lotus 1 2-3 program and the CSS statistics software packages. We obtained information on the size of the stained areas of the pars distalis (PD) in 139H-infected hamsters and on the optical density of these areas. This information was stored, analysed statistically and compared by Student's t-test with the data from sections obtained from animals inoculated with NHB homogenates. Results
Histopathology The pituitary gland consists of three parts, namely the pars nervosa (PN), pars intermedia (PI) and pars distalis (PD) (Figs 1 and 2). In control hamsters, the cells in the PD were closely packed together, forming thick branching cords of cells with numerous sinusoids (Si) located between them; occasionally, endothelial cells could be observed at the margins of the sinusoids. In 139Hinfected hamsters, there was extensive extracellular vacuolation in the pituitary glands. Most vacuoles were located in the ventral or ventrolateral parts of the PD. The pituitary cells were dispersed and the sinusoids damaged, especially in the area of vacuolation. In contrast, there was comparatively little vacuolation in the pituitary glands of 263K-infected hamsters (Figs 2b and 2d). As shown in Figs 1 and 2, the pituitary glands of 139H-infected hamsters also showed cellular hypertrophy, cellular atrophy and necrosis, and cytoplasmic vesicles. Nuclear changes included swelling, the appearance of ring-forms, vesicle formation, pyknosis, karyorrhexis and karyolysis. The cellular and nuclear pathological changes were most pronounced in regions with vacuolation. In contrast, there were comparatively few cellular changes in the pituitary glands of 263K-infected hamsters (Figs 2b and 2d). Use of Gomori's
Pituitary Changes in Scrapie .e
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"
--
i ~
Fig. 1.
~
-
.~
a-d. Haematoxylin and eosin stain of pituitary glands, a, b, Control hamster; c, d, 139H-infected hamster, a, x 65, b d, x 262. (PN, pars nervosa; PI, pars intermedia; PD, pars distalis). In the control hamster, the pituitary cells in the PD are closely packed with m a n y sinusoids (Si) located between them. However, in the 139H-infected hamster, the pituitary cells in the PD are more dispersed and many Si are destroyed. There are numerous vacuoles (*) located on the ventral or ventrolateral parts of the PD. Pituitary glands of 139H-infected hamsters show cellular hypertrophy and necrosis (arrowhead). Nuclear pathological changes are present, such as swelling (small arrow) and ring-form changes (medium arrow); vesicular changes, pyknosis, karyorrhexis and kar,volysis (arrowhead) are also evident. The cellular and nuclear pathological changes are most pronounced in the regions with vacuolation.
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Fig. 2.
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~ d . Pituitary glands in 139H- and 263K-infected hamsters stained with Gomori's one-step trichrome stain; a, c, 139H-infected hamster; b, d, 263K-infected hamster, a, b, x 321 c, d, x 262. (PI, pars intermedia; PN, pars nervosa; PD, pars distalis). There is extensive extracellular vacuolation (*) and cellular atrophy (arrowhead) in the ventral and ventrolateral parts of the PD in the 139H-infected hamster, with comparatively little vacuolation in the 263K-infected hamster. No abnormal collagen fibres are visible in the pituitary glands of these scrapie-infected hamsters.
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one-step trichrome failed to show any abnormal collagen fibres in the pituitary glands of 139H- or 263K-infected hamsters. Both Congo red and thioflavinS stains failed to show evidence of amyloid formation.
Immunocytochemistry To unmask epitopes of the aggregated abnormal isoform of the scrapie protein, sections were treated with 88% formic acid for 15 min before being allowed to react with the primary antiserum for PrP so. With this technique we previously demonstrated PrP sc formation and amyloid plaques in the cortex, hippocampus and substantia nigra, and around the pia mater, corpus callosum, fimbria, ventricles and blood vessels in sections from 263K-infected animals (Ye et al., 1993). In the present study, PrP sc immunolabelling was not seen in the pituitary glands of scrapie-infected or control animals. To investigate the cell types affected by scrapie infection, the immunolabelling patterns of several hormones were examined in the pituitary glands of control and 139H- and 263K-infected hamsters (Figs 3-5). In control animals, cells giving a positive reaction for ACTHI_24, ACTHI7 39, GH, T S H and PRL were distributed throughout the PD. The intensity of the immunostaining varied, even in sections stained with the same primary antibody. Some of the positive cells were located around the sinusoids. As shown in Figs 3 and 5, the immunostaining patterns of ACTHl_24 did not differ dramatically in 139H-infected, 263K-infected and control animals, positively reacting cells being found in the PI and PD but not in the PN. However, there were fewer ACTH1 24-immunostained cells in the extracellular vacuolation areas of the PD of 139H-infected hamsters than of other animals (Figs 3b and 3d). Control and 139H-infected hamsters differed slightly in respect of the pituitary immunostaining patterns of T S H (Figs 4 and 5) and PRL (not shown). TSH-immunostained cells were found primarily in the PD, with a few positive cells in the PI and none in the PN of either control or scrapie-infected hamsters. PRL-immunostained cells were found only in the PD. The TSHand PRL-immunostained cells were fewer in the PD vacuolation areas of 139H-infected hamsters than in control animals. As shown in Figs 4 and 5, the GH immunostaining patterns were similar in all three groups of hamsters. GH-positive cells were found mainly in the PD, but a few were seen in the PI, and immunostained fibres were seen in the PN. GH-immunostained cells occurred less frequently in the ventrolateral area than in other parts of the PD; since this was the area that showed the most extensive histopathological changes, it was not surprising that the effect on G H immunostaining in 139H-infected animals was minimal (Fig. 4 and Table 1). The immunostaining patterns of ACTHl_24, ACTH17 ~9, G H and T S H in the pituitary glands of 263K-infected hamsters are shown in Fig. 5. Vacuolation was not present and the patterns of these hormones and PRL were similar to those seen in the controls. There were only a few FSH- and LH-immunostained cells in the pituitary
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Fig. 3.
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~ d . Immunostaining patterns of ACTttj_24 in the PD of pituitary glands, a, Control hamster; b, c, d, 139H-infected hamster, a, b, x 65, c, x 131, d, x 262. The immunostaining ofACTH~ 24 is distributed throughout the pars distalis (PD) and pars intermedia (PI) in both the control hamster and the 139H-infected hamster. Arrowhead shows intensive ACTHI 24 immunostaining (ir-ACTHI 24) in PD and weak staining in PI. The number of cells stained for ACTHI-24 was reduced in those focal areas (*) that had extensive vacuolation in the 139H-infected pituitary gland.
Pituitary
Fig. 4.
Changes
in Scrapie
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~ d . Immunostaining patterns of T S H and GH in the PD of pituitary glands, a, x 65, TSHimnmnostaining (ir-TSH), control hamster; b, x 131, ir-TSH, 139FI-infected hamster; c, x 65, GH-immunostaining (ir-GH), control hamster; d, x 131, ir-GH, 139H-infected hamster; (PI, pars intermedia; PD, pars distalis; *, vacuolation area). The immunostaining of T S H and GH is distributed throughout the PD in the control hamster and the 139H-infected hamster. Arrowheads show T S H and G H immunostaining in PD and PI. GH-immunolabelled cells are more rare in the ventral and ventrolateral area than in other parts of the PD in both control and 139H-infected hamsters.
300
Fig. 5.
X . Ye a n d R . I. C a r p
a d. Immunostaining patterns of ACTHl-24, ACTHI7 39, G H and T S H in the pituitary glands of 263K-infected hamsters, a, ACTHI 24; b, ACTI-II7_39; C, GH; d, TSH; ~ d , x 65. (PI, pars intermedia; PD, pars distalis). The pituitary glands of 263K-infected hamsters do not show vacuolation in the PD as is seen in 139H-infected hamsters. Arrowheads show immunostaining in PD and PI. The immunostaining patterns with different antisera are very similar in the pituitary glands of the control and 263K-infected hamsters (Fig. 3a, Fig. 4a, c). The immunostaining patterns of A C T H I 24 and ACTHI7 ~9 are very similar to each other in both PD and PI, which suggests that both of the antisera stained cells containing full length A C T H .
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Pituitary Ghanges in Scrapie Table 1 I m m u n o s t a i n i n g of the pituitary glands o f 139H-infected and control h a m s t e r s Inoculum
Number of hamsters
NHB 139H NHB 139H NHB 139H NHB 139H NHB 139H NHB 139H NHB 139H
Immunostain target (hormone*)
10 11 11 14 15 13 15 18 11 15 12 15 13 14
Immunostaining results (+_ SEM) OD
ACTH~7 39 " ACTH~ 24 " GH " TSH " PRL " FSH " LH "
0"30 0"30 0'33 0'34 0'27 0"30 0'25 0"27 0"20 0'19 0'13 0'17 0'03 0"04
PA (mm 2)
(0"02) (0"01) (0"02) (0"02) (0"02) (0"02) (0"03) (0"02) (0"02) (0'02) (0'04) (0"02) (0'02) (0'02)
0"28 0'20 0"27 0"23 0'26 0"36 0"20 0"22 0'23 0"32 0'02 0"02 0"00 0"00
PA %
(0"03) (0"04) (0"03) (0"03) (0"04) (0"04) (0"02) (0"02) (0"06) (0"06) (0"01) (0"01) (0"00) (0"00)
11"9 9-6 13"1 11"6 11'1 13"0 9"0 9"4 10'9 14"2 0"9 1"2 0"l 0"0
(1"0) (1"6) (1'3) (1"3) (1"6) (1"7) (1"1) (0"8) (3'6) (2"6) (0"4) (0"5) (0"1) (0"0)
*Abbreviations defined in Materials and Methods. NHB, normal hamster brain; OD, optical density; PA, positively stained area of pars distalis; PA%, positively stained area expressed as a percentage of the whole pars distalis.
Table 2 Histopathological changes, infectivity titres and PrP concentrations in the pituitary gland and pancreas of 263K- and 139H-infected h a m s t e r s Organ
Pituitary gland Pancreas~
Histopathological changes
Infectivity titre* (mean)
PrP concentration
263K
139H
263K
139H
262K
139H
+
+ + +
6"3
8"8
ND
ND
-
+ + +
>3"9<5"4
>3'9<5"4
Low
High
* Log~0. "~Results from the studies of Carp et al. (1990, 1994) and Ye et al. (1994a). ND, not done; + + + , striking; _+, slight; - , absent.
glands of scrapie-infected and control animals. Most of the LH-positive cells were located at the edge of the ventral PD (not shown). Pituitary immunostaining results obtained from 139H-infected and control hamsters were compared with regard to optical density and to the positively stained area expressed as a percentage of the whole PD (Table 1). The results showed no significant differences (unpaired t-test; P>0"05). However, as noted above, there was a reduced number o f A C T H - , TSH- and PRL-immunostained cells in the focal areas of vacuolation seen in 139H-infected hamsters. Table 2 summarizes the data now available on the effects of 263K and 139H on the pituitary gland and pancreas. In the pituitary gland the 139H strain differed from the 263K strain in producing more severe histopathological changes and higher infectivity titres.
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Discussion The pituitary gland is structurally and functionally complex, controlling many other endocrine glands in the body. The synthesis and secretion of many pituitary hormones, such as PRL, FSH, LH, A C T H and GH, are influenced by a variety of stimuli (e.g., sleep, stress) and are episodic and pulsatile. Plasma concentrations of such hormones can change dramatically within minutes. This is especially true in females, because of the oestrous cycle. This may explain the high degree of variation in immunostaining patterns seen within single groups of the female hamsters used in these studies (Table 1). It seems possible that the obesity and hyperinsulinaemia induced in hamsters by the 139H strain of scrapie may be mediated by organs such as the hypothalamus or pituitary. In a previous study, 139H-infected hamsters showed a significant increase in neurons staining for a corticotropin-releasing factor (CRF) in the preoptic nucleus and a Significant decrease in neurons staining for vasopressin (VP) in the lateral hypothalamus (Ye et al., 1994c). The hypothalamus is an important control organ for the neuroendocrine system. Hypothalamic hormones are released from hypothalamic nerve fibre endings around capillaries in the pituitary stalk and reach the anterior lobe (pars distalis) through the special portal system. In addition, hypothalamic-releasing factors can stimulate the synthesis and release of pituitary tropic hormones, which can regulate the activities and functions of other endocrine organs. Both CRF and VP can stimulate the release of A C T H from the pituitary gland. Abnormal CRF and VP activity is capable of disturbing pituitary cellular functions and morphology. Abnormal changes in the islets of Langerhans, such as hypertrophy and hyperplasia of B cells, may be directly responsible for the obesity and hyperinsulinaemia reported in 139H-infected hamsters (Ye eta[., 1994a, b). The cause of lesions of the islets of Langerhans may be "local" (originating within the cells of the islets) or "central" (changes in the brain). Scrapie strain 139H can induce different pathological patterns in different organs of the hamster (Carp et al., 1993, 1994). Since scrapie infectivity titres in these organs (e.g., pancreas and adrenals) are low, it is unlikely that the scrapie agent directly causes pathological changes in organs outside the central nervous system. Such changes are probably induced indirectly and are a function of the specific "targeting" effect of the 139H strain in the central nervous system (Carp et al., 1990). If the lesions of the islets of Langerhans are based "centrally" on lesions in the brain, two possible pathways should be considered: hormonal and neuronal. In the neuronal pathway, lesions in the hypothalamus would stimulate the motor neurons of the vagus in the brain stem, increasing parasympathetic activity. Acetylcholine released from the vagus nerve can stimulate B-cell activity, and insulin-secretion-promoting factor released from the ventral hypothalamus can stimulate insulin release (Jeanrenaud, 1985; Bobbioni-Harsch and Jeanrenaud, 1989). An anterior pituitary hormone, galanin, can inhibit insulin secretion (Kaplan et al., 1988). In 139H-infected hamsters these hormones may be affected by the cellular damage in the hypothalamus and pituitary gland.
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Hamsters infected with the 139H scrapie strain showed extensive vacuolation in the pituitary glands, while the glands in 263K-infected hamsters showed little or none. There is a significant difference in incubation period between the two scrapie strains (139H, 135 days; 263K, 65 days). We do not know whether this difference could account for the more pronounced pituitary lesions in the 139H-infected hamsters. The different phenotypes seen in hamsters infected with one or other of the two scrapie strains (Carp et al., 1994), and the pathological changes seen in the pituitary glands of 139Hinfected but not 263K-infected hamsters, suggest that these two scrapie strains have different effects on either brains cells or pituitary cells. In the pituitary gland, strain 139H, unlike 263K, produced striking histopathological changes and high infectivity titres (Table 2), but neither strain gave rise to demonstrable degrees of amyloid formation or PrP sc accumulation. The degree of PrP sc accumulation in the pituitary glands was clearly less than that seen in the brain of scrapie-infected hamsters; infectivity titres were also lower in the pituitary gland than those that occur in the brain, particularly in 263Kinfected animals. In conclusion, this study supports a possible role of a hypothalamic or a pituitary factor in mediating the obesity and hyperinsulinaemia produced by infection with the scrapie 139H strain.
Acknowledgments We thank Drs A. Scallet, J. Fikes, and B. Bolon for critical reading of this manuscript. X. Ye was supported in part by the New York State Department of Mental Hygiene, in part by NIH grant AG0917, and in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. References Bobbioni-Harsch, E. andJeanrenaud, B. (1989). The hypothalamic origin of an insulin secretion promoting factor present in the plasma of normal rats. Journal of Neuroendocrinology, 1, 103 108. Carp, R. I., Kascsak, R.J. and Rubenstein, R. (1993). Pathogenesis of unconventional slow infections. In: Light and Electron Microscopic Neuropathology of Slow Firus Disorders, P. P. Liberski, Ed., CRC Press Inc., Boca Raton, Florida, pp. 33 61. Carp, R. I., Kim, Y. S. and Callahan, S. M. (1990). Pancreatic lesions and hypoglycemia-hyperinsulinemia in scrapie-injected hamsters. Journal of Infectious Diseases, 161,462 466. Carp, R. I., Ye, X., Kascsak, R. J. and Rubenstein, R. (1994). The nature of the scrapie agent: biological characteristics of scrapie in different serapie strain host combinations. In: Slow Infections of the Central Nervous System, J. Bjornsson, R. I. Carp, A. L6ve, and H. M. Wisniewski, Eds, Annals of the New York Academy of Sciences, 724, 221 234. Chandler, R. L. (1963). Experimental scrapie in the mouse. Research in Veterinary Science, 4, 276-285. Dickinson, A. G. (1977). Scrapie in sheep and goats. In: Slow Virus Diseases of Animals and Man, R. H. Kimberlin, Ed., North-Holland, Amsterdam, pp. 209 241. Diedrich, J. F., Bendheim, P. E., Kim, Y. S., Carp, R. I. and Haase, A. T.
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(1991). Scrapie associated prion protein accumulates in astrocytes during scrapie infection. Proceedingsof the National Academy of Sciences USA, 88, 375 379. Jeanrenaud, B. (1985). An hypothesis on the aetiology of obesity: dysfunction of the central nervous system as a primary cause. Diabetologia, 28, 502-513. Kaplan, L. M., Gabriel, S. M., Koenig, J. I., Sunday, M. E., Spindel, E. R., Martin, J. B. and Chin, W. W. (1988). Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proceedingsof the National Academy of Sciences USA, 85, 7408-7412. Kascsak, R. J., Rubenstein, R. and Carp, R. I. (1991). Evidence for biological and structural diversity among scrapie strains. Current Topics in Microbiology and Immunology, 172, 139-152. Kimberlin, R. H. and Walker, C. A. (1986). Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly. Journal of General Firology,67, 255-263. Prusiner, S. B. and DeArmond, S.J. (1994). Prion diseases and neurodegeneration. Annual Researchof Neuroscience, 17, 311-339. Ye, X., Carp, R. I., Kascsak, R., Kozielski, R. and Kozlowski, P. (1993). Astrocytosis and amyloid deposition in scrapie-infected hamsters. In: Biology and Pathology of Astrocyte-Neuron Interactions, S. Fedoroff, B. H. J. Juurlink and R. Doucette, Eds, Plenum Press, New York and London, p. 443 (Abstract). Ye, X., Carp, R. I., Yu, Y., Kozielski, R. and Kozlowski, P. (1994a). Histopathological changes in the islets of Langerhans in 139H-infected hamsters. Journal of Comparative Pathology, 110, 153-167. Ye, X., Carp, R. I., Yu, Y., Kozielski, R. and Kozlowski, P. (1994b). Hyperplasia and hypertrophy of B-cells in the islets of Langerhans in hamsters infected with the 139H strain of scrapie. Journal of ComparativePathology, 110, 169 183. Ye, X., Carp, R. I., Yu, Y., Kozielski, R. and Kozlowski, P. (1994c). Effect of infection with the 139H scrapie strain on the number, area and/or location ofhypothalamic CRF- and VP-immunostained neurons. Acta Neuropathologica, 88, 44-54.
Received, September8th, 1995] Accepted, December4th, 1995 J