Received for publication Sep 5, 1989; accepted Feb 28, 1990. Reprint requests to (R.L.L.) National Institutes of Health, Bldg 3, Room 106, Bethesda, MD 20892.
PEDIATRICS (ISSN 0031 4005).
A Difference
in Mortality
Between
Two
Strains
of Jaundiced
Rats
Paul E. Stobie, MD; Carl T. Hansen, DVM; James R. Hailey, DVM; and
Rodney L. Levine, MD, PhD
From the Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, and the Veterinary Resources Branch, National Institutes of Health, Bethesda, MD; the Walter Reed Army Institute of Research, Washington, DC; and the Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda
ABSTRACT. Homozygous Gunn rats lack bilirubin
glu-curonyltransferase, become jaundiced, and often develop
kernicterus, thus providing a model for neonatal
hyper-bilirubinemia. Two new, inbred rat strains that carry the
Gunn mutation are described. These were developed by
breeding the mutant Gunn gene (j) into the RHA/N and
ACI/N strains, producing the new lines, which were
designated RHA/N-j and ACI/N-j. Liver assay confirmed
the absence of transferase activity in jaundiced rats from
both of the new strains, but marked differences in
mor-tality between the strains were observed. The mortality
ofjaundiced RHA/N-j rats through 8 weeks was the same
as that of their nonjaundiced littermates (20%). In
con-trast, mortality of jaundiced ACI/N-j rats was distinctly
greater than that of their nonjaundiced littermates (81%
vs 34%, P < .001). Signs of kernicterus such as ataxia
were much more frequent in jaundiced ACI/N-j rats than
in jaundiced RHA/N-j rats (73% vs 11%, P < .001). Both
strains had comparable albumin concentrations through
8 weeks of age. Serum bilirubin concentrations were also
comparable, except for a small but significant difference
at 20 days of age (ACI/N-j = 294 mol/L, RHA/N-j =
248 mol/L, P < .01). Similarly, the bilirubin-to-albumin
ratios were comparable except for a significantly higher
ratio at 20 days of age in the ACI/N-j rats (ACI/N-j =
0.70, RHA/N-j = 0.51, P < .01). Thus, the RHA/N-j
strain is unusual in that the jaundiced animals remain
healthy. Conversely, the ACI/N-j animals demonstrate a
high incidence of kernicterus with mortality. This
dra-matic strain difference implies that factors in addition to
deficiency of bilirubin glucuronyltransferase are
modu-lating the susceptibility to bilirubin toxicity. Pediatrics
1991;87:88-93; bilirubin, kernicterus, Gunn rats, jaundice.
Hyperbilirubinemia is the most common medical
problem of the newborn. Despite its frequency, the
factors that affect the development of kernicterus
in the nursery or the laboratory remain elusive.’3
The Gunn rat has been studied as an animal
model of neonatal hyperbilirubinemia and
kernic-terus for decades. The homozygous Gunn rat lacks
bilirubin uridine
diphosphate-glucuronyltransfer-ase (EC 2.4.1.17), the enzyme necessary for
conju-gation of bilirubin to glucuronic acid.#{176}8The
enzy-matic defect is the same as that in the human
Crigler-Najjar type I syndrome. Developmental
im-maturity of this same enzyme is believed to
con-tribute to idiopathic or “physiologic” jaundice in
the newborn.9 Because the Gunn rat cannot
conju-gate bilirubin to glucuronic acid, very little bilirubin
is excreted in bile. Consequently, these animals
develop indirect hyperbilirubinemia, may
demon-strate neurologic abnormalities including
kernic-terus, and die.’#{176}’1
The Gunn mutation was discovered
serendipi-tously in a breeding stock of Wistar rats at the
Connaught Laboratories4 and has been introduced
into colonies around the world o that the “Gunn
rat” is not a well-defined congenic strain (personal
communication from Dr L. Johnson). The gene
responsible for jaundice in the Gunn rat is
desig-nated
j,
and J denotes the normal gene. To developa genetically defined and stable congenic strain of
jaundiced rats, we bred the Gunn rat’s
j
gene intotwo established inbred rat strains, the RHA and
ACI strains. From the outset we noticed an
appar-ent difference in mortality between the jaundiced
rats of these two new strains. Understanding the
basis for this difference should provide insight into
the toxicity of bilirubin. We therefore undertook a
prospective study of 500 rats from these two new
jaundiced rat strains. We now report striking
dif-ferences in mortality and incidence of kernicterus
Female (jJ) x male
(jj)
Female (jJ) x male (jJ)38 18
10.4± 2.8 6.1 ± 3.1
41% [1611
(ii)
24% [26] (ii)48% [1901 (jJ) 66% [73] (jJ or JJ)
11% [45J 10% [11]
METHODS
Strain Development
The mutant gene,
j,
was introduced into the ACI/N and RHA/N rat strains, two inbred lines
main-tamed at the National Institutes of Health since
1951 and 1968, respectively.’2 To establish the
RHA/N-j strain, a heterozygous (jJ) Gunn rat was
mated with an RHA/N rat. The Gunn rat
contrib-utes 50% of the genetic makeup of the resulting
offspring. This and each following generation was
thus composed of two genotypes, the heterozygous carriers
(jJ)
and normal homozygotes (JJ). The phenotype of both was nonjaundiced. Thehetero-zygotes could be identified by their capacity to
produce jaundiced rat pups
(jj)
when mated with a known carrier of thej
gene. Heterozygotes wereused for further breeding, allowing transmission of
the
j
gene to future generations. The continuedback-breeding of each generation of heterozygotes
with RHA/N rats progressively decreased the Gunn
rat’s contribution to the genetic makeup of each
new generation. This was repeated for 12
genera-tions, culminating in the new strain RHA/N-j. This
strain was congenic (99.98%) with the RHA/N
strain, except for the
j
gene. The ACI/N-j strainwas developed in the same fashion. Further breed-ing of both new strains were brother-sister matings,
ensuring an inbred, genetically stable population.
Detailed studies of glucuronyltransferase activities in the RHA/N strain have been reported.’3
Study Animals
Heterozygous RHA/N-j females (genotype
jJ)
were mated with homozygous RHA/N-j males
(ii)
with the expectation of 50% of the offspring being
jaundiced
(jj).
The poor survival of homozygous ACI/N-j males(if)
necessitated matingheterozy-gous ACI/N-j males
(jJ)
with heterozygous ACI/N-f
(jJ)
females, so that 25% of the offspringshould be jaundiced. Dams and litters were housed
in a 25#{176}Cwindowless room with overhead 12-hour on/off fluorescent lighting. Water and standard rat
chow were provided ad libitum. Pups were weaned
from their mothers at 27 to 28 days. Selective toe
clipping allowed identification of each pup,
permit-ting tracking of weights, clinical observations, and
laboratory studies. Observations were made daily
for the first 3 weeks of life, then spaced
progres-sively during the fourth to eighth weeks.
Fifty-six litters were studied as summarized in
Table 1. Ten percent of each strain were stillborn
or died before an accurate assignment of phenotype.
These nonphenotypeable pups were excluded from further analyses. The proportion ofjaundiced pups
encountered in the litters was consistent with au-tosomal recessive inheritance as established by
Gunn.4 Growth curves for each strain were
essen-tially the same as those shown by Gunn.5
Laboratory Values
Blood drawings from individual rats were usually
spaced 7 days apart during the first month of life
to avoid excessive losses. Blood was collected into
capillary tubes after tail-cut. Animals were killed
by decapitation after carbon dioxide
anesthetiza-tion, and then blood was collected either by cardiac
puncture or directly after decapitation. All blood
and sera were shielded from light. Serum was
sep-arated by centrifugation and stored at -20#{176}C.
Ex-cised brains were stored in light-protected formalin
before sectioning. Excised livers were weighed and
then immersed in an ice-cold solution of 250 mmol/
L sucrose and 1 mmol/L
ethylenediaminetetraace-tate, pH 7.4. Small sections from different lobes
were combined, minced, and homogenized in four
volumes of buffer using a Teflon piston rotating at
1300 rpm for 8 passes, then stored at -70#{176}C.
Con-trol experiments established that
glucuronyltrans-ferase activity was stable when prepared and stored in this way.
Serum bilirubin was assayed under low-light
con-ditions with first-derivative multicomponent
spectrophotometry’4”5 (Hewlett-Packard model
8450, Palo Alto, CA). Serum albumin level was
measured using a bromcresol green linked
spectro-TABLE 1. Characteristics of the Rat Strains
Characteristic RHA/N-j ACI/N-j
Parents’ genotypes No. of litters
Litter size* (mean ± SD)
Phenotypet Jaundiced Nonjaundiced Not determined
* The litter sizes differed significantly (P < .001).
t The number of animals is given in brackets.
100
80
60
40
20
0 0
Fig 1. Mortality of the four groups of rats. Mortality of
the RHA strain was not affected by the presence or
absence ofjaundice. Mortality was markedly increased in
the ACI strain when the animals were jaundiced (P < .01
by life-table analysis).
AGE, DAYS
photometric assay.’6 Activity of hepatic bilirubin
glucuronyltransferase was measured in the
homog-enates from 79 individual animals using a
modifi-cation of a diazo linked spectrophotometric assay,’7
and total protein concentration was determined
with a Coomassie blue assay.’8 The enzyme assay
was modified to include 5 mg/mL cetyltrimeth-ylammoniumbromide in the organic solvents just before the final centrifugation. The presence of this
cationic detergent facilitated the sedimentation of cellular debris and yielded a clear supernatant. Con-trol livers were from adult ACI/N rats, the
non-jaundiced parent stock of the ACI/N-j strain.
Pathology
Gross and microscopic examinations of the brains were performed by a single pathologist (J.R.H.), who was “blinded” to the strain and phe-notype of the submitted specimens. Animals were killed at ages 4 through 352 days. According to the
prospective design ofthis study, animals found dead were not included in the analysis, to avoid con-founding by postmortem changes. Representative levels of cerebral cortex, thalamus, hippocampus, brainstem, and cerebellum were studied in hema-toxylin- and eosin-stained slides. Cerebellar Pur-kinje cell layer lesions were scored as the percentage
of affected cells, notably cytoplasmic vacuolar
changes or necrosis. Two hundred cells were ex-amined in each cerebellum.
Statistical Analysis
Mortality and the incidence of neurologic
symp-toms were evaluated using life-table analyses.
Com-parisons between strains were analyzed with the two-tailed Student’s t test or the Fisher exact test
as appropriate. Data analyses were run on IBM
personal computers using Blossom templates for
the Lotus 1-2-3 program (B. R. Cole, Data Man-agement Branch, Division of Computer Research and Technology, National Institutes of Health). Fisher exact tests were calculated with the Epistat program written by T. L. Gustafson (Round Rock, TX). Results are stated as the mean ± sample standard deviation, unless otherwise indicated.
RESULTS
Mortality
Life-table mortality data are shown in Fig 1. The 8-week mortality rate of the jaundiced RHA/N-j rats was the same as that of their nonjaundiced littermates (20%). However, the mortality of the jaundiced ACI/N-j rats was significantly greater
than that of their nonjaundiced littermates (81 vs
32%, P < .01).
Clinical Signs and Symptoms
Jaundice was easily appreciated within the first
day of life in rat pups from both strains. Jaundiced
rats who died after 2 weeks of age characteristically
lost weight, appeared weak, and developed ataxia.
The rear legs would weaken, splay outward from
the body, and sometimes simply be dragged behind
the body. Loss of truncal support resulted in the
body’s being carried low to the ground. Rear support
was often impaired, giving the rats a hunched
pos-ture. Ataxic behavior was frequent. We observed
slow, uncoordinated walking, wobbly gait,
persev-erant turning in circles, stumbling, and falling
sideways in the days before death. Further
weak-ness progressing to an inability to stand generally
preceded death. Jaundiced ACI/N-j rats exhibited
such behavior much more frequently than their
RHA/N-j counterparts (73% vs 11%, P < .001).
Mean age of onset of symptoms was 18 days in both
strains, with a range of 7 to 31 days. Death was
inevitable when such severe signs persisted. As
observed by Gunn4’5 and later workers, the severe
ataxia in the affected animals prevented them from taking food and water in the day(s) before death.
Thus, changes in behavior and biochemical values
in the day or so before death may result from
malnutrition rather than from the direct effect of
the Gunn mutation. Those jaundiced animals that
survived generally did not demonstrate
abnormali-ties, except that the majority ofthe jaundiced RHA/
N-f pups transiently exhibited rapid to-and-fro
L1 c’
30 AGE, DAYS
observed in the other animals and was not predic-tive of later ataxia or mortality. Seizure activity,
opisthotonos, and hypertonicity were seen only
rarely. However, daily observations were brief and
these could have been missed.
Hepatic Glucuronyltransferase Activity
Given the dramatic differences in mortality
be-tween the two new strains, it seemed important to confirm the lack of bilirubin uridine
diphosphate-glucuronyltransferase. Specific activities were
de-termined in liver homogenates from 73 rats (Fig 2).
The jaundiced phenotype was specific for
glucuron-yltransferase deficiency, with essentially no
detect-able activity. In 21 jaundiced rats (19 RHA/N-j, 2
ACI/N-j) the mean specific activity was 2 pmol/
mm per milligram of protein (median = 1). Fifty of
their nonjaundiced littermates averaged 198 ± 46 pmol/min per milligram of protein, and the two samples from the parental strain RHA/N averaged 369 pmol/min per milligram of protein.
Serum Bilirubin Concentration
Mean serum bilirubin concentrations of the jaun-diced rats during the first 8 weeks of life are shown
in Fig 3. The pattern is the same as that reported by Johnson and colleagues,’#{176} although her values
were somewhat lower than ours, probably because of differences in the assay for bilirubin. Serum
bilirubin levels were comparable between the
strains, except for a small but statistically
signifi-cantly higher level in the ACI/N-j pups at 20 days (294 ± 14 vs 248 ± 33 mol/L, P < .01). On day 22
z
TRANSFERASE ACTIVITY
Fig 2. Distribution of hepatic bilirubin uridine
diphos-phate-glucuronyltransferase specific activities. The units
of activity are picomoles per minute per milligram of protein. All jaundiced rats had essentially no detectable activity (<25 pmol/min per milligram of protein). None
of the nonjaundiced rats fell in that range.
a single ACI/N-j bilirubin concentration was
meas-ured, but it was not significantly different from that
of nine RHA/N-j bilirubin concentrations.
We compared serum bilirubin levels of
nonsur-viving jaundiced rats with those of surviving
jaun-diced rats. Although nonsurvivors tended to have
higher bilirubin levels at approximately 3 weeks of
life, there were no statistically significant
differ-ences. Serum bilirubin levels of rats with and
with-out kernicteric symptoms were also compared,
ir-respective of strain. A trend toward higher bilirubin levels for the kernicteric animals was observed at approximately 3 weeks of age, but again this was
not statistically significant.
Serum Albumin Concentrations
No differences were found when serum albumin
concentrations were compared between jaundiced and nonjaundiced rats of each strain (Fig 4). Serum albumin level increased during the first 4 weeks of
life, then plateaued at approximately 500 mol/L
(3.3 g/dL). Thus, the bilirubin-to-albumin ratios
were similar for both strains, except for a
signifi-cantly higher ratio at 20 days of age in the
ACI/N-j
rats (0.70 ± 0.026 vs 0.51 ± 0.055, P < .01). Thisdifference was due to the higher bilirubin concen-tration on that day, as noted above.
Fig 3. Mean serum bilirubin concentration of the
jaun-diced rats. Closed circles represent the ACI/N-j animals
and open circles represent the RHA/N-j animals. The
age, number of animals, and the standard deviation for
the RHA strain were, respectively: 1, 8, 22; 2, 8, 33; 3, 11,
31; 5, 14, 53; 8, 15, 71; 10, 13, 23; 12, 10, 44; 13, 16, 27;
16, 12, 37; 18, 12, 35; 20, 15, 33; 22, 9, 52; 24, 17, 45; 28, 17, 63; 32, 17, 23; 36, 11, 38; 40, 14, 33; 52, 8, 24; 57, 24,
69. For the ACI animals the age, number of animals, and
standard deviation were: 4, 6, 89; 5, 3, 79; 6, 2, 36; 8, 4,
25; 13, 10, 24; 20, 5, 14; 22, 1, NA; 28, 2, 18; 36, 1, NA;
600
500
400
z 300
200
100
30 AGE, DAYS
Fig 4. Mean serum albumin concentrations. No differ-ences were found between the albumin concentrations of
the jaundiced and nonjaundiced rats in each strain. Thus,
results from both phenotypes were plotted in the figure.
Closed circles represent ACI animals and open circles represent RHA animals. The age, number of animals,
and the standard deviation for the RHA strain were,
respectively: 1, 10, 0.4; 2, 7, 0.2; 3, 23, 0.2; 5, 24, 0.5; 8, 21, 0.2; 10, 21, 0.3; 12, 15, 0.3; 13, 25, 0.1; 16, 23, 0.2; 18, 20, 0.2; 19, 5, 0.1; 20, 28, 1.1; 22, 8, 0.2; 23, 2, 0.1; 24, 29,
0.9; 27, 5, 0.1; 28, 29, 1.3; 31, 2, 0.3; 32, 21, 1.1; 36, 22, 1.0;
40, 13, 0.2; 41, 14, 0.3; 45, 4, 0.1; 51, 23, 1.4; 56, 55, 0.8.
For the ACI animals the age, number of animals, and
standard deviation were: 3, 1, NA; 4, 5, 0.2; 5, 4, 0.5; 6, 5,
0.3; 8, 5, 0.1; 9, 9, 0.2; 12, 6, 0.4; 13, 7, 0.2; 18, 3, 0.1; 20, 12, 1.2; 22, 5, 0.4; 24, 4, 0.3; 26, 2, 0.1; 28, 6, 0.4; 30, 4, 0.1; 36, 8, 0.2; 38, 2, 0.3; 45, 1, NA; 55, 4, 0.1; 56, 12, 0.1.
Pathology in the RHA Strain
A total of 48 brains were studied from RHA rats killed at 4 to 352 days. A similar study of the ACI animals was not undertaken because ofthat strain’s high mortality. Comparisons were made between
jaundiced (n = 21) and nonjaundiced RHA rats (n
= 27). Attention was focussed on the Purkinje cells
of the cerebellum because they are most commonly affected in Gunn rats, as originally described by Schutta and Johnson.’9 Table 2 shows that Pur-kinje cell changes were significantly increased in the RHA/N-j animals compared with the non-jaundiced rats, although abnormalities occurred in fewer than half of the jaundiced animals. No differ-ences were observed in the cerebrum nor brainstem. Also, slides of liver and kidney from the same 48
animals revealed no substantive abnormalities.
DISCUSSION
Colonies of Gunn rats are maintained throughout the world, with varied breeding practices fostering the development ofdistinct subpopulations of Gunn rats (personal communication from Dr L. Johnson). Indeed, some Gunn rat colonies are pigmented, although Gunn’s original colony was albino. These
TABLE 2. Cerebellar Abnormalities in the RHA Strain*
Purkinje Cell Changes Phenotype
Jaundiced Nonjaundiced
Present, % [no.] 38 [8] 7 [2]
Absent, % [no.] 62 [13] 93 [25]
* Brains were assessed without knowledge of the animal’s
phenotype. Purkinje cell changes included cytoplasmic
vacuolization and cell necrosis and were scored as present
when seen in at least 5% of the 200 cells that were
assessed. The observed differences are significant (P =
.01 by Fisher’s exact test).
Gunn rat colonies lack genetic uniformity and may
not be comparable. Jaundiced Gunn rats have been
reported to have a 25% to 80% mortality rate, fluctuating with periods of intercurrent illness in the rat colonies.’0 Clinical or pathologic kernicterus
was evident in approximately 60%, and neurologic
sequelae were noted in 50% of the survivors.’0” Keino and colleagues dealt with the variability in their Gunn colony by introducing the
j
gene intothe Sprague-Dawley strain of rats. This congenic
strain had a mortality rate of 80% by 30 days of
age.2#{176}
The jaundiced rats of the ACI/N-j and
RHA/N-j
strains differed markedly in incidence of clinicalkernicterus and mortality. Glucuronyltransferase
deficiency was clearly associated with poor outcome in the ACI/N-j strain (46% attributable mortality
and 73% kernicterus), but not in the RHA/N-j strain (0% attributable mortality and 11%
kernic-terus). This disparate mortality and morbidity is
evidence for inherited factors, other than deficiency of the specific glucuronyltransferase, which
modu-late susceptibility to bilirubin toxicity. (We assume
that glucuronyltransferase deficiency is essential for poor outcome. However, one should note that other genes have been transferred from the Gunn
strain. As described in the Methods section, the
new strains are 99.98% congenic with the parental
strains. The residual 0.02% cannot be ignored. While unlikely, it is possible that the gene required for neurologic damage is closely linked to, but not identical with, the glucuronyltransferase gene.) Quantitative albumin levels were similar between the strains. Small but statistically significant dif-ferences in bilirubin levels and bilirubin-to-albumin ratios were apparent in the jaundiced ACI/N-j rats at 20 days of age. Thus, the difference in mortality
might be explained by differences in serum bilirubin concentrations, although those differences must be attributed to factors other than the mutant
glucu-ronyltransferase. As Odell and colleagues have
itself.2’ Indeed, alterations in any of the
compo-nents that regulate bilirubin production and
dis-posal could provide the basis for the difference in mortality that we observed.9’22
One should also note that 34% of the mortality attributed to the presence of the mutant
glucuron-yltransferase occurred before 20 days. Furthermore, 45% (5/11) of the symptomatic ACI/N-j rats de-veloped symptoms before 20 days. Examination of
the life table (Fig 1) for the ACI/N animals
con-firms that there are two periods of increased
mor-tality of the jaundiced ACI/N animals: an early
period (#%.5 to 14 days) and a late period (>24 days). The RHA/N-j and ACI/N-j strains are congenic with their parental strains, offering a defined and stable genetic background. Fortuitously, the mci-dence of symptomatology and mortality was polar-ized between the two new strains. The RHA/N-j
strain was little affected by jaundice whereas the
ACI/N-j rats were decimated. These rats and their parental strains thus provide an opportunity to investigate the determinants of bilirubin toxicity. (Animals will be made available to investigators
who wish to establish their own breeding colonies. Please write to Chief, Small Animal Section, Vet-erinary Resources Branch, National Institutes of Health, Building 14A, Room 103, Bethesda, MD
20892.) From the data presented here it is clear
that factors other than absence of glucuronyltrans-ferase must be important. It is of historical interest to note that Gunn himself seems to have reached a
similar conclusion. Furthermore, he hypothesized
that vitamin A might be the other factor. In his original report4 in 1938, he noted poor growth and
neurologic signs in the mutant animals and then
stated, “Experimental evidence indicates that the lag in growth and nervous symptoms are [due to] a prolonged vitamin A deficiency.” Six years later he published a paper that included data to support that statement.5 Some 50 years after the original
report, it still seems important to pursue the iden-tification of those other factors.
ACKNOWLEDGMENTS
While these studies were carried out, P.E.S. was a
Fellow in Neonatal Medicine at the Uniformed Services
University of the Health Sciences.
We thank Susan Turkington for technical assistance
with these studies.
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