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This is to certify that the

thesis entitled
Effects of Cisplatin on the Rat NeurohypOphysis
presented by

Philip J. Boyer

has been accepted towards fulfillment
of the requirements for

M.S. degreein Biology

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Date 11/14/86

 

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EFFECTS OF CISPLATIN ON THE RAT NEUROHYPOPHYSIS

BY

Philip Joseph Boyer

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Interdepartmental Biological Sciences Program

1986

‘l 97- 01" 3'1

ABSTRACT
EFFECTS OF CISPLATIN ON THE RAT NEUROHYPOPHYSIS
By
Philip Joseph Boyer

Cisplatin treatment (5, 7, or 9 mg/Kg) of young wistar rats
elilcits a dramatically decreased urine output and water
intake. Neurohypophysial examination at the light and
electron microsopic level either 3 or 5 days after drug
treatment reveals two distinct types of abnormal appearing
pituicytes (glial cells). 1.) Pituitaries from rats treated
with 7 mg/Kg cisplatin have pituicytes which accumulate
abnormally large amounts of glycogen and lipids. 2.)
Pituitaries from 9 mg/Kg treated rats have numerous
translucent-appearing pituicytes. Thus, cisplatin effects
on the neurohypophysis appear to be dose-related. While
these effects are similar to pituicyte changes elicited by
varying lengths of long-term water deprivation of rats, it
is concluded that cisplatin treatment affects rat
neurohypophyses in ways other than by physiologic

dehydration and may in fact have direct toxic effects.

ACKNOHLEDGMENTS

I wish to thank the following people:

-Dr. S.K. Aggarwal: for his optimistic support and guidance
of the various aspects of my research and scholarship; his
nearly limitless expertise and "magic" with histologic and
cytochemical techniques; his electron micrographs; and his

help with most every part of this research

-Jim Fadool: for his help with nearly every element of this

research and his thought-stimulating discussions

-Dr. P. Gotschall: for the numerous hours of help with my

incomplete vasopressin RIA work

-Mark Batzer: for his encourgement of the research and his

help with drug injections and animal sacrifices

-Dr. M.Z. Jones: for her analysis of the electron

micrographs

-Dr. N. Band: for his thoughtful criticism of the manuscript

TABLE OF CONTENTS

LIST OF FIGURES ..................................
INTRODUCTION....... .............................
MATERIALS AND METHODS ............................
RESULTS ..........................................
DISCUSSION .......................................

REFERENCES .......................................

25
31

LIST OF FIGURES

Figure Page
1 Rat Hater Intake and Urine Output ........... 12

2 Light Micrographs

A. Control.. ............................ I“
B. 7 mg/Kg Cisplatin, 5 days ............ ‘6
C. 9 mg/Kg Cisplatin, 5 days ............ 18

3 Electron Micrographs

A. Control .............................. 20
B. 7 mg/Kg Cisplatin, 5 days. ........... 22
C. 9 mg/Kg Cisplatin, 5 days ............ 24

INTRODUCTION

The antieneoplastic activity of platinum compounds was
first documented in 1969 (38). Cisplatin (cis-dichloro-
diammineplatinum(II)) is an effective chemotheraputic agent
in the treatment of human testicular (43,10) and ovarian
cancers (22) and produces significant results in the treat-
ment of cervical, bladder, prostate, and head and neck
(epithelial) cancers (11,15). Cisplatin's promising results
are hindered by a broad spectrum of side effects (7.24) of
which the most clinically important and dose-limiting is
renal toxicity (18,19). Hydration and diuresis therapy as
well as timing of treatments have decreased the severity of
renal toxicity (13.23.42.43) but high dose therapy still
risks considerable renal damage (6,33).

The mechanisms of Cisplatin's actions and related
toxicities and side effects are not yet clearly understood.
Cisplatin's major anti-neoplastic effects are thought to be
the result of inhibition of nuclear replication by inter-
strand DNA cross-linking (36,46). However, other actions
of the drug may be significant too. Cisplatin has been
shown. in vitro. to inhibit karyokinesis and arrest cyto-

kinesis (1,2). In addition, cisplatin both in_vivo and in_

 

vitro inactivates a variety of plasma membrane-associated
enzymes including adenosine triphosphatases. alkaline
phosphatase, and 5‘-nucleotidase (3.31). This enzyme
inactivation leads to cell permeability changes which can

alter cell function and may lead to cell death (5).

2

Much research has gone into the development of analogs
of cisplatin in an attempt to duplicate or improve on its
anti-neoplastic activity but decrease its side effects
(26.28.45). However. each analog has its own problems
with solubility. stability. activity. and toxicity (43.45).

Cisplatin is a proven. effective anti-neoplastic
drug. Because of its documented success with testicular and
ovarian tumors and its potentially broad application in the
treatment of other solid tumors (11.15.16) a more complete
documentation of cisplatin's side effects would be of
considerable significance. Knowledge of treatment's side
effects may help ellucidate its mechanism of cytotoxicity
and then lead to a means of limiting undesirable side
effects.

One side effect of cisplatin treatment in 18. 22. and
38 week old Wistar rats is a marked decrease in urine output
(8). While extensive renal tubule damage and decreased
glomerular filtration rate by the drug may alter water
metabolism (8.12.20). cytotoxic effects on the posterior
pituitary and then on the release of vasopressin may be
important as well. Various studies have examined
cisplatin's renal cytotoxicity (18.24) and some studies
have examined plasma vasopressin levels in cisplatin-treated
rats (12.20). but no reports of cisplatin treatment's
morphologic effect on the posterior pituitary are available.

In this study. the morphologic effects of cisplatin
treatment on the neurohypophysis were examined both to

evaluate potential cytotoxic or dehydration responses of the

3
organ and to provide a background for radioimunoassay (RIA)
and cytochemical studies of vasopressin in the drug—treated
rat. Such studies may help extend the current attempt at
delineation of water metabolism disturbances associated with

cisplatin treatment.

4

MATERIALS AND METHODS

Animal Preparation

Male Swiss Wistar rats (Crl:(WI)BR-—Charles River Breeding
Laboratories) weighing between 190 and 290 g and of 55-65
days in age were injected intraperitoneally with a single
dose of 5. 7. or 9 mg/Kg cisplatin (Johnson Matthey Research
Laboratories. Sonning. U.K.). The cisplatin was dissolved
in normal saline (0.75 M) and allowed to stand overnight in
a 35 C incubator. Controls recieved saline injections.
There were 20 rats in each experimental and control group.
All experimental procedures were performed between 9 am and
11 am during consecutive 24 h periods. The day of injection

of cisplatin or saline was called day 0.

Physiologic Studies

All drug-treated and control animals were provided. ad
libitum. with measured amounts of tap water and Wayne Lab-
Blox rat food (Allied Mills). Effective cisplatin treatment
in Wistar rats is characterized by significant. steady
weight loss and decreased physical activity. For urine
output assessment. control and cisplatin-treated animals
were placed for 24 h in metabolic cages to which they were
acclimated for 2-3 d prior to drug treatment. Water intakes
and weight changes were measured daily for a period of a

week prior to and during experimental studies.

Tissue Preparation
Experimental and control animals in each treatment group

were sacrificed by decapitation 3 days (10 rats) or 5 days

5
(10 rats) after drug or saline injection. Trunk blood was
collected in heparinized tubes. centrifuged. and volumes
were recorded. Pituitaries were quickly removed after
decapitation. sliced in half longitudinally. and one half of
each pituitary was placed in Hollande's Bouin (HB) for 4 h
for light microscopy while the other half was processed for
electron microscopy. HB-fixed tissues were dehydrated in
graded ethanol and embedded in Paraplast. 7 um sections
were cut using a rotary microtome (American Optical) and
processed routinely. Deparaffinized slides were stained
using the periodic acid-Schiff technique and counterstined
with Ehrlich hematoxylin (25). Salivary diastase digestion
(30 min) prior to periodic acid treatment was performed on
negative glycogen control slides (25). For electron
microscopic studies. tissues were fixed in 1% osmium
tetroxide and 1% glutaraldehyde (both in 0.05 M sodium
cacodylate buffer at pH=7.2) for 3 h. dehydrated in a graded
acetone series. and embedded in epon. Thin sections were
cut on a LKB Ultratome III ultramicrotome and stained with

uranyl acetate and lead citrate.

Microscopy

PAS-hematoxylin stained sections were examined and
photographed using a Zeiss Photomicroscope 11. Thin sectins
were examined and photographed using a Hitachi HU11E
electron microscope Operated at 75 Kv. Sections from near
the center of the pituitary were used for all analyses. but

each pituitary was examined at various sequential levels of

sectioning for consistancy.

Data Analysis

Blood volumes were standardized by dividing total drained
trunk volume (ml) by body weight (Kg) and the percent
decrease of drug-treated rat blood volumes versus control
blood volumes were calculated. Urine output and water
intake volumes were standardized by dividing volume (ml) by
body weight (Kg) and graphed (Fig. 1) as mean +/- standard

deviation.

7

RESULTS

Physical. Physiologic. and Necropsy Observations

Cisplatin-treated rats at all dosage levels moved around
their cages much less frequently and vigourously than did
control rats. Rats treated with 7 or 9 mg/Kg of cisplatin
generally sat motionless in a corner of their cage. By day
5. rats in all dosage groups showed considerable cachexia.

A dramatic decrease relative to control volumes is noted
in both urine output and water intake at days 1 (not
shown). 3 and 5 after cisplatin injection at all dosage
levels (Fig. 1). Weight-standardized drained trunk blood
volumes decreased significantly (P 0.05) relative to
controls by 16%. 28%. and 31% for 5mg/Kg. 7 mg/Kg. and 9
mg/Kg cisplatin-treated rats. respecively. Blood from
drug-treated rats appears more viscous. especially at day 5
for the high dosages levels (7 and 9 mg/Kg). than the blood
of control rats. At necropsy. all cisplatin-treated rats
showed considerable bloating and ulcerations of their
stomach and their stomach contents were watery. Few to no
fecal pellets were noted in their intestines although a few
rats in the high dosage groups had developed diarrhea.
Control rats had non-bloated stomachs. moist but compact
stomach contents. and numerous fecal pellets in the

intestines.

Morphologic Findings
Figure 2 shows light micrographs of posterior pituitary

cross-sections taken from control (A). 7 mg/Kg (B) and 9

8

mg/Kg (C) cisplatin-treated Wistar rats 5 d after injection.
Figure 3 shows electron micrographs from the same dosage
groups. also 5 days after injection. With the exception of
small amounts of glycogen accumulation in a few peripheral
cells. no glycogen is seen in most control pituicytes (Fig.
2A). Virtually all control pituicytes have a dense.
basophilic-staining cytoplasm. Electron micrographs (Fig.
3A) of control pituicytes and axons shows large nuclei with
dispersed chromatin; normal and evenly distributed Golgi.
endoplasmic reticulum (ER). and mitochondria; no marked
glycogen accumulation; and a few lipid globuoles. In large
cross-sections of pituicytes a variable number of
cytoplasmic extensions can be seen which tortuously reach
out among. and sometimes encircle. axons. Axons are evenly
distributed and a large number of neurosecretory vesicles is
seen within them.

Neurohypophyses of 5 mg/Kg cisplatin-treated rats are
virtually indistinguishable morphologically from controls.
although a few pituicytes have clear cytoplasmic rings
surrounding their nuclei. Peripheral glycogen and lipid
accumulations are of the same order of magnitude as those in
control tissues. No apparent decrease in the number or
tortuosity of cytoplasmic extensions is noted.

Neurohypophyses from 7 and 9 mg/Kg cisplatin-injected
animals always show one of two distinct. abnormal patterns
of pituicyte appearance. At the light microscope level (Fig.
28). cross-sections of pituitaries from 7 mg/Kg treated

animals show striking accumulations of glycogen within some

pituicytes while other pituicytes have less or no
accumulation. The pituicyte glycogen granulation in this
group of pituitaries is most pronounced around the perphery
of the tissues and becomes less intense centrally. The
glycogen appears to be exclusively in pituicytes. Glycogen
accumulation is approximately of the same magnitude at 3 and
5 d in this group. Also. many pituicytes are seen to have a
clear ring of cytoplasm surrounding their nuclei. At the
electron microscope level (Fig. 38). large lipid globules
can be seen in cells which have very heavy glycogen
accumulations. Such cells tend to retain cytoplasmic
extensions among axons. However. the extensions reach
neither as far nor as tortuously as do those in control
tissues. The distribution of cytoplasmic organelles in
glycogen- and lipid-rich pituicytes appears normal although
ER and Golgi tend to have a somewhat swollen appearance.
Pituicytes loaded with glycogen and lipid can lie in close
proximity to a pituicyte with little to no glycogen buildup
and normal-appearing cytoplasmic organelles. Axons in this
group of pituitaries contain fewer neurosecretory granules
than do control axons. The clear cytoplasmic rings observed
at the light microscope level cannot be distinguished at the
electron microscope level.

At the light microsc0pic level, cross-sections from 9
mg/Kg cisplatin-treated pituitaries appear to have a third
or less of the amount of glycogen seen in 7 mg/Kg treated
pituitaries. but roughly three times the amount found in

control tissues. At 3 days after drug treatment virtually

l0
all pituicytes have a clear ring of cytoplasm. variable in
size. surrounding their nuclei. After 5 days between 15 and
25 percent of pituicytes per mid-pituitary section at low
power are seen to have a rounded-up appearing, translucent
cytoplasm while the rest of the pituicytes retain the
clear ring appearance. At the electron microscope level
(Fig. 30). cells with translucent cytoplasm are seen to have
few to no cytOplasmic extensions among axons and their
cytoplasm appears centralized. Heterochromatin is
marginated along intact nuclear membranes and clumped
centrally in these cells. These pituicytes' cellular
organelle content appears diminished and the organelles tend
to be clumped and appear swollen. Cytoplasmic membranes
are intact and no inflammatory cell infiltration is noted.
No vascular changes are seen. Lamellated-bodies. between 1
and 4 axons per total area of mid-pituitary sections are
seen. Most axons contain fewer neurosecretory granules than
do control axons. Non-translucent pituicytes have dispersed
chromatin and normal distribution of organelles. yet their
cytoplasmic extensions are less far-reaching than those seen
in control pituicytes. No cytoplasmic rings can be

identified at the EM level.

ll

Figure 1 Graph showing water intake and urine output in
control versus cisplatin-treated rats at various

dose levels 3 and 5 days after treatment

 

12

 

3; 0.3.

0.1-

 
  
   

 

 

. WATER INTAKE
URINE OUTPUT

DAY 3

 

s
S
R
\
N
\
N
S
S

o 7 ‘ 9“
CISPLATIN
TREATMENT (mg/Kg)

Figure 2 A Control Pituritary. Light micrograph showing
pituicyte nuclei (P). and endothelial cell

nuclei (E). PAS-Hematoxylin. 1800X.

 

 

15

Figure 2 B 7 mg/Kg Cisplatin Treated Pituitary. 5d. Light
micrograph showing pituicyte nuclei (P). clear
cyt0plasmic rings ( -+>). and glycogen (-* ).
PAS-Hematoxylin. 1800X.

 

Figure 2 C

17

9 mg/Kg Cisplatin-Treated Pituitary. 5d.

Light

micrograph showing translucent pituicytes (-+).

glycogen ( -+ ). and endothelial cell nuclei

PAS-Hematoxylin. 1800X.

(E).

 

l8

 

19

Figure 3 A Control Pituitary. Electron Micrograph showing
axons (A). pituicyte membrane (-9) and nucleus

(N). and lipid globules (L).

 

20

 

 

Figure 3 B

7 mg/Kg
Electron
membrane

globules

2l

Cisplatin-Treated

Pituitary. 5d.

micrograph showing pituicyte

(-O). glycogen (-0). lipid

(L). and axons (A).

17.200X

22

 

Figure

3

23

9 mg/Kg Cisplatin-Treated Pituitary. 5d.
Electron micrograph showing pituicyte nucleus
(N). translucent cytoplasm (C). and membrane
( -+). lipid globules (L). axons (A). and
lamellated body (8). 13.200X.

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1" T'
.v

2A

 

25

DISCUSSION

The highly vascular neurohypophysis consists of axons
arising from hypothalamic supraoptic and paraventricular
nuclei and of astrocytic glial cells called pituicytes. No
easy morphologic distinction exists between vasopressin and
oxytocin carrying neurons. Pituicyte cytoplasm is quite
extensive and cytoplasmic processes are typically seen. at
the electron microscope level, to reach out among and
sometimes surround axons in their vicinity in normal. well-
hydrated rat pituitaries (21.30.40). It is usually difficult
or impossible to trace the pituicyte cytoplasmic extensions
in an entire section of a cell because of their tortuous
interweaving among and between axons (Fig. 2A). The role of
the pituicytes in the neurohypophysis is not entirely clear.
They are thought to play an important role in the modulation
of vasopressin release (21.40). Further. they help in the
removal of necrotic axons (14.27.35) as well as in the
development of the neural lobe (17). They probably also play
a classic glilal support role in the maintenance of axonal
metabolism.

Cisplatin-treated rats are physiologically dehydrated
as demonstrated by an alteration of their water metabolism:
urine output and drained trunk blood volume are decreased
while blood viscosity is increased. A drug-induced
dehydration could be contributed to by a number of factors.
Most importantly. water intake is significantly decreased
beginning at day 1 and extending to day 5 after drug

treatment (Fig. 1). A protracted decrease in water intake

26

probably directly contributes to all other water metabolism
changes. In addition. it may be that only part of the
water that is taken in by drug-treated rats is effectively
absorbed. The stomachs of cisplatin-treated. Wistar rats.
at any of this study's dosage levels. undergo dramatic
bloating (4.37). The contents of these bloated stomachs are
considerably more watery than those of control rats and this
water retention may limit the amount of water that reaches
digestive tract absorptive surfaces. In addition. although
little food passes through the digestive tract of cisplatin-
treated rats (4) some 7 and 9 mg/Kg treated rats have a
slight degree of diarrhea which would lead to further water
loss.

Plasma vasopressin levels during cisplatin treatment
have been reported only in Sprague-Dawley rats. and only at
1 day (20) and between 8 and 24 h (12) after drug
injection. These rats undergo a diuresis and polydipsia in
response to cisplatin treatment. In contrast. cisplatin-
treated Wistar rats are dehydrated and vasopressin levels
would thus be expected to increase in them and would
contribute to a decreased urine volume and increased urine
osmolarity and thus conserve water. It is possible that
cytotoxic effects on pituicytes could elevate vasopressin
levels beyond what woud be expected for the particular
dehydration level present and depress urine output still
further. Radioimmunoassay studies of plasma vasopressin

levels in cisplatin-treated Wistar rats are forthcoming

27
from this lab. Damage to kidney tubules is severe following
single-dose cisplatin treatment at most dosage levels (5.19)
and may also contribute to a decreased urine output although
Sprague-Dawley studies cite renal damage as possibly leading
to increased urine output (18.24).

The distinct morphologic responses of pituicytes to
cisplatin treatment seems to be dose-related and may be
indicative of varying degrees of pituicyte response to
injury. Rats deprived of water for between 1 and 15 days
show moderate to heavy accumulation of glycogen and lipids
(29.32). This experiment's water deprivation studies. while
carried out for only 3 and 5 days. showed both pituicyte
glycogen and lipid buildups. considerably greater than
control levels. However. these levels of accumulation come
nowhere near the amount of the substances found in 7 and 9
mg/Kg cisplatin-treated rats. Large glycogen and/or lipid
accumulations are also found in a variety of physiologic and
experimental situations including during develOpment of the
neural lobe (17). during aging (35). after hypophysial stalk
transection (14.27) and after reserpine treatment of rats
(34). All of these reports claim that the appearance of
glycogen and/or lipid accumulations is indicative of the
"activation" of pituicytes involved with increasing the
amount of vasopressin released from axons or in stimulating
the growth of. maintaining. or phagocytizing axons (i.e.. in
support roles). At least for the water deprivation
experiments. a correlation exists between dehydration (which

should elicit increased vasopressin release) and glycogen

28

and lipid accumulation. The massive glycogen and lipid
accumulations seen in cisplatin-treated rat pituitaries may
be associated with an activation of the cells in response to
both dehydration and injury.

Electron micrographs of well-hydrated rat
neurohypophyses show a large percentage of axonal processes
to be in contact with or surrounded by a pituicyte's or
pituicytes' cytoplasm (40). The number of glial enclosures
of and contacts with axons is seen to be significantly
decreased by water deprivation of between 4 h and 4 d or by
increased osmotic pressure as induced by raising ionic
content in vivo or in vitro (21.40.44). The reduction of
pituicyte-axon contact is thought to result from retraction
of pituicyte cytoplasmic processes (41). The formation of
translucent pituicytes in 9 mg/Kg cisplatin-treated
pituitaries is probably a function of retraction of
cytoplasmic processes. but to a point much greater than is
elicited by the 4 h through 4 d water-deprivation. That
this is probably the case is supported by the finding of
numerous "degenerating" pituicytes in 15 day water-deprived
pituitaries (32). the electron micrographs of which are
indistinguishable from cisplatin-induced translucent
pituicytes.

The clearness of translucent cells may be due. at least
in part. to the dilutional effects which result from the
rounding up and centralization of these pituicytes'

cytoplasm. The clear rim of cytoplasm seen surrounding most

29

pituicyte nuclei in 7 and 9 mg/Kg cisplatin-treated rats.
only at the light microscopic level (Fig. ZB.C). may be
the result of the moderate cytoplasmic process retraction
noted in these cells.

The pituicyte changes seen in 7 and 9 mg/Kg cisplatin-
treated rats. like those seen in water deprivation studies.
may be distinct pituicyte responses to different levels of
cellular injury. With slight or moderate injury the
pituicyte may respond with varying degrees of cytoplasmic
process retraction and accumulation of glycogen and lipid.
With severe injury the pituicyte may respond by totally
retracting its cytoplasmic processes and accumulating some
lesser amount of glycogen and lipid. There may also be a
point where long-term. moderate injury culminates in total
retraction of cytoplasmic processes.

The mechanism of action of cisplatin which might lead
to the hypothesized pituicyte injury is not clear.
Cisplatin-induced dehydration cannot by itself account for
the rapidity or magnitude of change in water metabolism that
is seen. The neurohypophysial effects of cisplatin
treatment bring about morphologic reSponses similar to. but
more extensively and rapidly than. long-term water
deprivation. Relative to the anterior pituitary. cisplatin
treatment has been shown to decrease serum levels of the
hormones LH and prolactin in pregnant rats by 39% and 63%.
respectively (9). Cisplatin is not found to accumulate in
general brain tissue (39). but accumulation Specifically in

the hypophysis has not been investigated. The blood-brain

3O

barrier not withstanding. the degree of vascularity of the
gland suggests at least some contact with the drug is
probable. if only at low levels. but especially with high-
dosage cisplatin treatment. Cytotoxic effects would than
possible.

While the huge stores of glycogen and lipid in
otherwise healthy. although somewhat retracted. pituicytes
can almost certainly return to normal levels. it is not
clear whether the translucent cells are irreversibly injured
or whether they can recover with time. The translucent
cells are certainly damaged. however the integrity of the
plasma and nuclear membranes. the lack of pyknosis of the
nuclei. and the lack of inflammatory cell infiltration
indicate that such cells are not yet necrotic and that
recovery is possible. An examination of neurohypophyses
from 7 and 9 mg/Kg treated rats at 7. 10. 15. and 30 days
after treatment will be undertaken to watch the course of
both the glycogen and lipid accumulations and the

translucent cells.

REFERENCES

10.

11.

31

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