1021865

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VDIXXX10.1177/10406387211021865Autolysis and the postmortem interval in horsesWenzlow et al.

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Journal of Veterinary Diagnostic Investigation

Feasibility of using tissue autolysis to estimate 1­–9

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DOI: 10.1177/10406387211021865
https://doi.org/10.1177/10406387211021865
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Nanny Wenzlow,1 Dan Neal, Adam W. Stern, Dhani Prakoso, Junjie J. Liu,
Gretchen H. Delcambre, Sally Beachboard, Maureen T. Long

Abstract. Estimation of the postmortem interval (PMI) is a poorly studied field in veterinary pathology. The development
of field-applicable methods is needed given that animal cruelty investigations are increasing continually. We evaluated
various histologic criteria in equine brain, liver, and muscle tissue to aid the estimation of PMI in horses, which is central
to forensic investigations of suspicious death. After death, autolysis proceeds predictably, depending on environmental
conditions. Currently, no field-applied methods exist that accurately estimate the PMI using histology in animals or humans
through quantification of autolysis. Brain, liver, and skeletal muscle from 12 freshly euthanized horses were held at 22°C
and 8°C for 72 h. Tissues were sampled at T0h, T1h, T2h, T4h, T6h, T12h, T24h, T36h, T48h, T60h, and T72h. For each
tissue, we quantified 5 to 7 criteria associated with autolysis, based on the percentage of microscopic field involved. Each
criterion was modeled, with temperature and time as independent variables. Changes were most predictable in liver and
muscle over the first 72 h postmortem. The criteria for autolysis that were present most extensively at both temperatures
were hepatocyte individualization and the separation of bile duct epithelium from the basement membrane. The changes
that were present next most extensively were disruption of myofiber continuity, hypereosinophilia, and loss of striation.
Brain changes were highly variable. The high statistical correlation between the parameter “autolysis” and the variables
“time/temperature”, indicates that autolysis is progressive and predictable. Further investigation of these criteria is needed
to establish histologic algorithms for PMI.

Keywords: autolysis; horses; postmortem interval; veterinary forensic pathology.

After death, the blood supply to tissue compartments is
compromised, causing acute hypoxic cell injuries that
manifest as acute cellular swelling, hydropic and vacuo-
lar degeneration, reduction in adenosine triphosphate
(ATP), membrane damage, increased intracytoplasmic
calcium, and formation of reactive oxygen species, all
leading to cell death and initiation of autolysis (self-
digestion).8,9 After death, this process of cell death is
postmortem autolysis, the earliest step of decomposition,
which is a process of denaturation of intracellular pro-
teins and nucleic acid, protein coagulation, and enzy-
matic digestion of nuclei and cytoplasm (Table 1).
Enzymes are released postmortem from the swollen lyso-
somes of dying cells by diffusion through the lysosomal
membrane.9,11 More specifically, the enzymes driving
autolysis are mostly phosphorus-rich enzymes including
alkaline and acid phosphatases, ATP, 5′-nucleotidases,
and glucose-6-phosphatase.11,18
Histologically, the morphologic changes of cell autolysis
are similar, if not identical, to those of cells undergoing necro-
sis, which is focal-to-extensive, non-programmed cell death
that occurs after irreversible exogenous injury to a cell (Table
1). Necrosis is also a process of progressive enzymatic cell
death, but within living tissue, and denaturation of intracellular
proteins is caused mostly by lysosomal enzymes released from
immigrant leukocytes, which are major actors in any inflam-
matory process.8 Necrosis can be initiated by a lack of blood
supply as described above, as well as by infectious agents
(bacteria, viruses, parasites, and protozoans) or toxins, which
all, more often than not, cause inflammation.6,8,9,13,19,20
A third type of cell death is apoptosis, a programmed pro-
cess that occurs most often in healthy tissues and is consid-
ered a physiologic event in which unwanted cells are
eliminated but can occasionally be pathologic after some
forms of cell injury (Table 1).8 Detailed descriptions of autol-
ysis and necrosis are available in the literature.8,10
Using light microscopy, necrosis and autolysis have simi-
lar morphologic changes, except that autolysis occurs without
Department of Pathology and Microbiology, University of Montreal,
Montreal, Quebec, Canada (Wenzlow); Departments of Surgery (Neal)
and Comparative, Diagnostic, and Population Medicine (Stern, Prakoso,
Liu, Beachboard, Long), University of Florida, Gainesville, FL, USA;
Department of Biomedical Sciences, Colorado State University, Fort
Collins, CO, USA (Delcambre).
1
Corresponding author: Nanny Wenzlow, Department of Pathology
and Microbiology, University of Montreal, 3200 rue Sicotte, St
Hyacinthe, Canada QC J2S 2M2. wenzlown@gmail.com
2 Wenzlow et al.
Table 1.  Comparison of the features of autolysis, necrosis, and apoptosis.
Autolysis
Occurs in living tissue No
Occurs only postmortem Yes
Programmed cell death No
Occurs in healthy tissue Yes
Physiologic process NA
Lethal injury necessary No
Accompanied by inflammatory reaction No
Enzymatic digestion Yes: enzymes released from
the dying cell itself
Harmful effects on surrounding tissue Yes
Caused by infectious or toxic agents No
NA = not applicable.
inflammatory cell infiltrates. Autolysis is also seen in the
entire organ or body, occurring usually at the same rate, and
occurs in tissues that have not been fixed immediately after
the patient’s death. Cells or tissues placed immediately in
fixative are dead but not necrotic, if fixation is prompt and
adequate.8,9 Focal cell death observed adjacent to normal-
appearing tissue in the same section suggests that this cell
death is necrosis and not postmortem autolysis.9 Unfortu-
nately, autolysis can be present in an irregular and patchy dis-
tribution. Furthermore, red blood cells often appear hemolyzed
in autolyzed tissues, suggesting a postmortem event.8,9
Necrotic tissue present in tissue that has not been fixed rap-
idly after death will also undergo postmortem autolysis.9 Dif-
ferent cell types undergo postmortem autolysis at different
rates because certain cells are more resistant to hypoxia than
others.1–3,5,8 The intestinal mucosa, gall bladder, pancreatic
parenchyma, and adrenal medulla autolyze first, then neurons
are the next most susceptible, with connective tissue the slow-
est to undergo degradation.1–3,5,8,10 Given that much of this
activity is enzymatically driven, the rate of autolysis as well
as its evolution to bacterial putrefaction, is slowed by refrig-
eration.
Histologic changes of autolysis during the early postmor-
tem interval (PMI) result from alteration of cell membrane
permeability brought about by ischemia and cessation of
metabolic activity.8,9 Autolysis can resemble necrosis. Necro-
sis is commonly accompanied by other pathologic changes,
including a visible inflammatory reaction and/or formation
of coagulative material, liquefaction, mummification, case-
ous debris, mineral deposits, and fibrin.6,8,13,19,20 Inflamma-
tion does not occur in autolysis. Autolytic changes are very
familiar to histopathologists, and these changes can interfere
with accurate histologic evaluation. Criteria are limited for
quantifying autolysis and hence to estimate the time of
death.7,11,12,15–17 Postmortem changes in the liver have been
described in guinea pigs.16 Neuronal changes in humans are
described in a handful of studies.11,12,15 Autolytic postmortem
changes have been described in postmortem traumatized
skeletal muscle in dogs17 and humans.7 Autolytic changes are
Necrosis Apoptosis
Yes Yes
No No
No Yes
No Yes
No Yes
Yes No
Yes No
Yes: enzymes released by No
surrounding leukocytes
Yes No
Yes Yes
not currently used by medical examiners to declare time
since death in humans (personal communications). Impor-
tantly, to our knowledge, very limited work describes quan-
titatively the progression of the intensity and distribution of
autolytic changes in any species.
Historically, autolysis has been associated with time since
death,8 given that it progresses continuously over time; how-
ever, histologic evaluation has not been used to systemati-
cally quantify histologic changes over exact time intervals,
especially in species that are common subjects of forensic
investigations. One such species commonly investigated is
the horse because of the value placed on sport, performance,
and breeding. Although forensic analysis of suspicious
equine deaths is a common event as a result of insurance
claims, malicious treatment, theft, and overall value of these
animals, horses are rarely the subject of forensic research.
However, time of death, an essential component of each
investigation, remains a challenge to estimate if the death
was not witnessed (e.g., euthanasia).
We investigated the hypothesis that specific histopatho-
logic changes associated with autolysis would occur in a
time- and temperature-dependent manner in equine tis-
sues. Our major aim was to perform a systematic analysis
of changes associated with autolysis of equine tissues. Tis-
sues from 12 horses were utilized to achieve a power of
≥0.80, and liver and muscle were chosen based on publica-
tions demonstrating that liver was least stable whereas
muscle was most stable over time.8 Brain was included
because there was little information regarding degradation
of brain during PMI and, given the large size of the horse
brain, we wondered if autolysis could be more predictable
in this species.
More often than not, the literature describes the use of a
limited number of or only one animal, limited sample times
used for analysis, and variable length of analysis as a result
of insufficient tissues being available.14,17 We designed our
study to provide a more comprehensive analysis of autolysis
in equine tissues. We quantified histomorphologic criteria of
autolysis in the equine brain, liver, and skeletal muscle during
 Autolysis and the postmortem interval in horses 3

Table 2.  Signalment of horses used in our study of autolysis. Histopathology and tissue criteria
Horse Sex Age (y) Weight (kg) The slides of each tissue held at each temperature were
blinded and read twice on a light microscope (DMLS Dual
1 M 21 U
2 F 12 U
VI; Leica) by one of the authors (N. Wenzlow), a trained
3 M U 496 veterinary pathologist. All slides from fresh tissues at T0h
4 M U 473 were evaluated for the absence of antemortem tissue necro-
5 M 19 570 sis or other lesions that might interfere with the interpreta-
6 F 15 510 tion of autolysis in tissue samples from later times (Suppl.
7 M 22 485 Fig. 1).
8 U U 464 Criteria of autolysis (Suppl. Table 1) were selected based on
9 U 15 510 literature review.7,11,12,14–18 Five representative fields per slide
10 M 4 340 were evaluated at 200× magnification for each criterion of
11 M 4 470 autolysis. In each field, the presence or absence of each crite-
12 F 11 590 rion was recorded and graded in the following 6 categories:
grade 0 if a criterion was absent, grade 1 if a criterion was pres-
F = female; M = male; U = unknown.
ent in <20% of the field, grade 2 if a criterion was present in
21–40% of the field, grade 3 if a criterion was present in 41–60%
the first 72 h (3 d) of the PMI. Criteria were based on changes of the field, grade 4 if a criterion was present in 61–80% of the
described previously.8,11,18 field, and grade 5 if a criterion was present in 81–100% of the
field.
Materials and methods
Statistical analysis
Study design
The grading of all 5 fields was averaged for each slide. Then,
Twelve healthy horses (Table 2) without clinical signs, in
the grades from all horses were averaged for each criterion for
adequate body condition (adequate subcutaneous and
each time. Data were grouped by tissue and temperature and
intra-abdominal adipose tissue), surrendered by owners
treated as non-repeated measurements. For all analyses of the
because of loss of use, were euthanized using sedation
histologic criteria, the data were graphed to examine trends
with a combination of acepromazine HCl (2–4 mg/45 kg)
over time for each criterion. If a pattern was not discerned,
and xylazine (0.5 mL/45 kg) intravenously, followed by an
correlation between time and outcome was determined using
overdose of pentobarbital and phenytoin (10 mL/45 kg,
the Spearman rank correlation coefficient. When a pattern
Beuthanasia-D; Merck Animal Health). Each animal was
was apparent, linear, parabolic, square root, or exponential
declared dead once ocular and palpebral reflexes ceased.
equations were derived depending on the shape of the graph.
All animal work was performed with approval of the Uni-
The best-fitting line was modeled, and a p-value calculated
versity of Florida Institutional Animal Care and Use Com-
using R v.3.2.4 (https://www.r-project.org/).
mittee (201004309).
After exsanguination, a large sample (10 × 20 × 20 cm) of
semitendinosus muscle, another large sample (20 × 20 × 5 cm) Results
of liver, and the entire brain were removed from each car-
Liver
cass. Each tissue was bisected, and one-half was maintained
at ~22°C, the measured room temperature of the autopsy In samples held at 22°C, we noted linear increases in hepatic
facility; the other half was maintained at 8°C, the measured plates separating from one another, leaving clear spaces
temperature of the autopsy walk-in cooler. The organs were between the rows of hepatocytes (hepatocyte individualiza-
kept in large, sterilized containers and covered with alumi- tion, p ≤ 0.0001; Fig. 1), bile duct epithelial cell separation
num foil. Tissues were sampled for histopathology at “time from the basement membrane (p = 0.0001; Fig. 2), and bile
0 hour” (T0h), T1h, T2h, T4h, T6h, T12h, T24h, T36h, duct epithelial pyknosis (p = 0.030; Fig. 3).
T48h, T60h, and T72h. The time between death and T0h In samples held at 8°C, we noted increases over time in
was 40 min for all horses. Brain samples were taken only variably sized, irregular empty vacuoles in hepatocellular
from the cerebrum, starting at the rostral lobe, throughout cytoplasm (hepatocyte vacuolation, p = 0.02; Suppl. Fig. 2)
the entire cerebrum to the caudal lobe, which provided the and bile duct epithelium separation from its basement mem-
T72h samples. A 0.5-cm slice of each tissue was placed in a brane (p = 0.0003; Suppl. Fig. 3). The correlation for hepato-
tissue cassette and fixed for 24 h in 10% neutral-buffered cyte vacuolation was best fit as the square root over time; the
formalin, followed by immersion in 75% ethanol. Formalin- bile duct epithelium separation from its basement membrane
fixed tissues were processed routinely, and 4-µm sections increased linearly over time. For all remaining criteria, there
stained with hematoxylin and eosin. was no significant trend over time.
4 Wenzlow et al.

Figure 1.  Hepatocyte individualization in the livers of 12 horses held at 22°C over 72 h postmortem. A. Each point is the average field
involvement of the criterion in 5 fields at 200× magnification at each time (0 = absent, 1 = 1–20%, 2 = 21–40%, 3 = 41–60%, 4 = 61–80%),
p < 0.0001. B. Hepatocyte individualization (arrows) in a horse liver at 60 h. H&E. 200×. Inset: hepatic plates separated from each other. 400×.

Figure 2.  Bile duct epithelium separation from its basement membrane in the livers of 12 horses held at 22°C over 72 h postmortem.
A. Each point is the average field involvement of the criterion in 5 fields at 200× magnification at each time (0 = absent, 1 = 1–20%, 2 =
21–40%), p = 0.0001. B. Bile duct epithelium separation from its basement membrane (arrows) in a horse liver at 60 h. H&E. 400×.

In the liver of 4 horses (22, 21, 19, and 15 y old), there was mixed-cell inflammatory infiltrates (Suppl. Fig. 4). This was
mild inflammation independent of autolysis and consisting considered an incidental, age-related finding, and did not
of minimal-to-mild, mostly portal, and occasionally random, interfere with our evaluation of autolysis.
 Autolysis and the postmortem interval in horses 5

Figure 3.  Bile duct epithelium pyknosis in the livers of 12 horses held at 22°C over 72 h postmortem. A. Each point is the average field
involvement of the criterion in 5 fields at 200× magnification at each time (0 = absent, 1 = 1–20%), p = 0.0001. B. Bile duct epithelium
pyknosis (arrow) in a horse liver at 72 h. H&E. 200×.

Skeletal muscle
For skeletal muscle maintained at 22°C, we noted linear
increases over time of shortened myofibers that left empty
spaces, referred to as interruption of fiber continuity
(p = 0.013; Fig. 4A, 4C), increased myofiber eosinophilia
(p = 0.003, Fig. 4B, 4C), loss of striation (p = 0.003; Fig.
5A, 5C), and a floccular and granular aspect of myofibrils
referred to as sarcoplasmic fragmentation (p = 0.046; Fig.
5B, 5C).
For skeletal muscle maintained at 8°C, myofiber eosino-
philia increased linearly (p ≤ 0.0001) and loss of striation
(Suppl. Table 1) initially increased, peaked at T48h, and then
decreased slowly, consistent with a significant parabolic cor-
relation with time (p = 0.010). For all remaining criteria,
there was no significant trend over time.
The criterion of segmental hypercontraction of skeletal
muscle fibers or large-diameter dark fibers was absent in all
examined sections of muscle from all horses at all times.
Brain
For brain maintained at 22°C, neuronal nuclear swelling
decreased initially over time, with its lowest point occurring
at T36h, before increasing (Suppl. Fig. 5A). This was statisti-
cally significant (p = 0.005), in a decreasing parabolic rela-
tionship with time. Vacuolation of the neuropil (Suppl. Fig.
5B) occurred in a significant (p = 0.004) nonlinear trend with
time, increasing initially and peaking at T24h, and then
decreasing, and was best fit as the square root over time.
Hence, nuclei initially shrink then swell, while at the same
time, vacuolation of the neuropil occurs and then regresses.
Although these criteria were statistically significant, the per-
centage of field involved was 20–40%.
At 8°C, neuronal pyknosis (p = 0.039) as well as Nissl
substance dissolution (p = 0.012) increased significantly as
a linear trend over time (Suppl. Fig. 5C, 5D). Neuronal
cytoplasmic swelling (Suppl. Fig. 5E) exhibited a signifi-
cantly decreasing (p = 0.014) linear relationship with time,
suggesting continuous shrinkage. Vacuolation of neuropil
(Suppl. Fig. 5F) exhibited a nonlinear trend, with the best
fit line corresponding to a square-root relationship with
time (p = 0.012). Although these criteria were statistically
significant, the percentage of field involved was 24–40%.
Neuronal cytoplasmic vacuolation was absent in all
examined sections of brain from all horses at all times. We
found that sex and age (young vs. adult) were not signifi-
cant variables.
Discussion
We observed the most consistent and significant changes at
22°C in liver and muscle tissue, with criteria observed in
>80% of the fields over time. Brain was the least predict-
able at either temperature. Most of the significant criteria
showed linear correlations with time, whereas others dem-
onstrated a significant parabolic, square-root, or exponen-
tial trend. For most of the nonlinear relationships, limited
field involvement was observed, making these parameters
unreliable. Occasionally, outliers were reported at single
times, which were considered incidental and independent
6 Wenzlow et al.

Figure 4.  Interruption of myofiber continuity and myofiber eosinophilia in semitendinosus skeletal muscle of 12 horses held at 22°C
over 72 h postmortem. A. Each point is the average field involvement of the criterion in 5 fields at 200× magnification at each time (0 =
absent, 1 = 1–20%, 2 = 21–40%), p = 0.013. B. Each point is the average field involvement of the criterion in 5 fields at 200× magnification
at each time (0 = absent, 1 = 1–20%), p = 0.013. C. Interruption of myofiber continuity (arrows) and skeletal muscle fiber hypereosinophilia
(*), in horse skeletal muscle at 72 h. H&E. 200×.

of autolysis. We observed that all histologic changes started
early after death and evolved with time; no criteria appeared
with delay at a later time.
In liver, individualization at 22°C changed the most
extensively, becoming apparent in ≥80% of the fields
examined at 72 h. This criterion was followed by bile duct
epithelium separation from the basement membrane, and
within bile duct epithelial pyknosis. In skeletal muscle, loss
of striation at 22°C occurred in up to 60% of the fields
examined by 72 h.
In a study16 of the sequence and rate of autolytic changes
of hepatocytes and bile ducts over 48 h in guinea pig livers
maintained at 22°C, hepatocellular cytoplasmic basophilia
was lost completely by 3 h, with the cytoplasm staining more
eosinophilic. No changes in hepatocyte basophilia were sta-
tistically significant at either temperature in our examined
equine livers. Guinea pig hepatocyte cytoplasmic vacuola-
tion and granularity increased progressively from 9 h through
36 h, when in horses, vacuolation was only significant at 8°C
and observed as early as 1 h after death, and was evolving
following a square-root relation with time over 72 h. Nuclear
fading and chromatin margination was apparent at 9 h, and
after 48 h, ~25% of the guinea pig hepatocyte nuclei had
lysed. Hepatocyte chromatin margination in horses was not
statistically significant at either temperature over 72 h.
Individualization of hepatocytes following dissociation
of single or groups of hepatocytes was first noticed at 6 h
and was most extensive at 48 h, when 25% of hepatocytes
were affected in guinea pigs. Individualization of equine
hepatic plates was observed as early as 1 h after death and
was most extensive at 72 h, when up to 80% of hepatocytes
were affected. In portal areas, the separation of bile duct epi-
thelium from the basement membrane was first observed at
18 h in guinea pigs and was present in most bile ducts by
24 h. Bile duct epithelium separation from the basement
membrane in horses was the only criterion observed with
statistical significance at both temperatures, as early as 1 h
postmortem and was most significant by 72 h at 22°C,
 Autolysis and the postmortem interval in horses 7

Figure 5.  Myofiber loss of striation and sarcoplasmic fragmentation in semitendinosus skeletal muscle of 12 horses held at 22°C over
72 h postmortem. A. Each point is the average field involvement of the criterion in 5 fields at 200× magnification at each time (0 = absent, 1 =
1–20%, 2 = 21–40%, 3 = 41–60%), p = 0.003. B. Each point is the average field involvement of the criterion in 5 fields at 200× magnification
at each time (0 = absent, 1 = 1–20%), p = 0.046. C. Loss of striation (*) and sarcoplasmic fragmentation (arrow) in horse skeletal muscle at
72 h. H&E. 200×.

affecting up to 30% of the bile ducts. Chromatin margin-
ation of guinea pig bile duct epithelial nuclei was first seen
at 6 h, and by 48 h most bile duct epithelial cell nuclei were
affected; chromatin margination in bile duct epithelial nuclei
was not significant at either temperature in horses.
Another study18 described progressive hepatocellular
pyknosis as well as hepatocyte individualization over 6 h of
postmortem autolysis in the rat liver, which is similar to the
progressive hepatocyte individualization seen in our study.
Significantly contrasting results were seen in dog liver deg-
radation.1 Bile duct epithelium detachment from the base-
ment membrane occurred at day 3 in dogs but was detected
after 1 h in our horses. By day 7, most canine hepatocyte
nuclei were autolyzed, and the most significant hepatocyte
autolysis was observed at 3 wk. Although our study did not
extend past 72 h, one could speculate that, by 3 wk, hepato-
cytes would also show the most significant autolysis. This
illustrates that liver changes are clearly not comparable
between species, and criteria should be established on a
species-by-species basis. Those differences may be the
result of differences in the size of animals, skin thickness,
and hair coat, which affects the body cooling rate.
Autolytic changes occurring in skeletal muscle are less
well studied. In one of the first studies, human skeletal muscle
tissue was electrically stimulated after death7 to affect changes
over time. In another study, changes were studied in canine
skeletal muscle traumatized after death followed by exposure
to sea water during the decomposition process.17 Changes
consisted of interruption of individual skeletal muscle fiber
continuity with loss of cross-striation (rupture of vital fibers),
multiple interruptions of fiber continuity with formation of
separate groups of sarcoplasm with loss of cross-striation
(segmental disintegration of fibers), and disc-like separation
of sarcoplasm with interruption of fiber continuity (discoid
disintegration of fibers). In comparison, we observed the
most significant changes in myofibers in horses at 22°C, and
loss of striation was the only criterion observed significantly
at both temperatures.
In general, most of the changes that we observed in the
cerebrum were subtle and were not present in large per-
centages of the fields examined. Brain autolysis in humans
showed neuronal Nissl substance dissolution, and karyor-
rhexis progressively increased from 5 to 22 h postmor-
tem.12,14,15 Nissl substance dissolution in horses was only
significant at 8°C, was seen as early as 1 h, and increased
linearly until 72 h. Neuronal cytoplasmic vacuolation,
basophilic cytoplasmic staining, and nuclear and cytoplas-
mic swelling were also considered to be autolytic changes
in humans. In horses, neuronal nuclear swelling was only
significant at 22°C; cytoplasmic swelling was only significant
8 Wenzlow et al.
at 8°C, and neuronal cytoplasmic vacuolation was not sta-
tistically significant at either temperature. Autolytic
changes in the human brain were first discernable as swell-
ing of the neuronal nucleus and cytoplasm with increasing
chromatolysis and liquefaction of the cytoplasm that may
or may not involve the nucleus.14 These initial changes
appeared at different times after death,11 with observations
occurring as early as 30 min or as late at 3 h postmortem.
Such chronology of events was not observed in equine
brains; all autolytic changes in the brain were subtle, and
no single most significant criterion stood out as a major
marker of autolysis.
We observed a striking difference between the intensity
and distribution of autolysis between the tissues kept at
22°C compared to the ones kept at 8°C (Suppl. Table 1).
This was particularly evident in muscle tissue in which
most criteria were significant at 22°C and not at 8°C, and
in the case of loss of striation, its highest field representa-
tion was 60% at 22°C, and was only 10% at 8°C. Similar
observations were made for the criteria in liver tissue, in
which bile duct separation from the basement membrane
was seen in 30% of the fields at 22°C, and in only 12% of
the fields at 8°C. Hence, autolysis was both slowed with
cooling and progressed as a different trend over time. In
the liver, hepatocellular individualization, vacuolation,
and bile duct epithelium pyknosis changed the most
between temperatures and were only significant at 22°C.
In brain and skeletal muscle, pyknosis, Nissl substance
dissolution, neuronal nuclear and cytoplasmic swelling,
interruption of myofiber continuity, and sarcoplasmic frag-
mentation of skeletal muscle cytoplasm were the most
variable between temperatures. Some changes were not
present at all or only present at 22°C by 72 h after death,
and these included hepatocellular chromatin margination
and basophilia, and margination of chromatin in bile duct
epithelium nuclei.
Although the inter-horse variation could have been
reduced by analyzing samples from the same horse for all
times, a study involving separate animals was necessary to
establish intra-horse variation based on age and sex that
could be applied to future statistical models using the criteria
found most reliable in our study.
Separation of tissues from each carcass was one limita-
tion of our study; thus, our data do not reflect field condi-
tions. Given the size of the horses, storage of 12 carcasses
was not feasible. Most of the criteria that we studied could
foreseeably be less reliable when analyzed in the context of
a full carcass for several reasons. For instance, it would be
expected that maintenance of vascular connections between
organs would allow migration of gut microbiota to liver,
which would accelerate putrefaction. Organs within the
body would likely maintain higher temperature initially,
allowing longer protein and enzyme activity of any process
of degradation and therefore increase the rate of autolysis.
The chosen temperatures in our study were 22°C and 8°C,
which were the autopsy room and walk-in cooler tempera-
tures.
Our equine candidates were selected based on their over-
all good health, adequacy of nutritional condition, and with a
wide age range. With increasing age, there may be underly-
ing conditions that our study did not account for. These may
include geriatric (suboptimal vascular circulation) or neo-
plastic conditions that alter metabolic rates affecting lyso-
somal protein degradation during autolysis. Variation in
body temperature could also play a major role in influencing
protein or enzyme activity, whereby patients dying with
fever would see an increased progression of autolysis versus
patients dying in hypothermia. These factors may all affect
decomposition rate and, if there is antemortem tissue necro-
sis, autolysis would be difficult to assess accurately. Gross
postmortem analysis should allow one to discern necrosis
from autolysis based on lesion location and distribution.
Autolysis will affect the entire body, and an entire organ
more uniformly, which microscopically will be seen in an
entire section of tissue.
Given that our data were analyzed as non-repeated mea-
surements, only correlation of trends with time could be
established. This design and analysis lack confidence inter-
vals (CIs) for the measures of central tendency and predic-
tive value. Therefore, the results of our study are not ready to
be used to estimate the postmortem interval in field cases.
There may be a rough indication of PMI based on the extent
of distribution in observed fields of each criterion. By
increasing the number of subjects with repeated measure-
ments of these significant criteria for autolysis, it could be
possible to establish regression equations with CIs for the
mean and possibly predictive values. If this is possible, then
histologic criteria could be used to estimate the PMI as well
as a 95% CI for the estimate of the time of death.
Taking the results from our study and what is known from
the literature, it seems that criteria of autolysis should be
established separately for each species. Multiple temperature
levels with longer times could provide data that lay the
groundwork for establishing PMI criteria that are more
objective than current parameters such as body temperature.4
Investigation of additional tissues will provide an extensive
and detailed picture of how autolysis affects tissues after
death and could possibly be used to estimate the PMI.
Acknowledgments
We thank Drs. Nancy Denslow, DeEtta Mills, Jason Byrd, Michael
Warren, and Martha Burt for their assistance with this project.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
 Autolysis and the postmortem interval in horses
Funding
Our research was funded by the Fern Audette Endowment in
Equine studies, University of Florida Graduate Fellowship Award,
8. Kumar V, et al. Robbins and Cotran Pathologic Basis of
the Emerging Diseases and Arbovirus Research Laboratory
(EDART), College of Veterinary Medicine, University of Florida,
9. Jones TC, et al. Veterinary Pathology. 6th ed. Lippincott,
and the American Society for the Prevention of Cruelty to Ani-
mals grant 2013-0107.
10. Miller MA, Zachary JF. Mechanisms and morphology of cellu-
ORCID iDs
Nanny Wenzlow https://orcid.org/0000-0003-3105-8267
Adam W. Stern https://orcid.org/0000-0002-6794-6592
Supplemental material
Supplemental material for this article is available online.
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