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Brief communication State of preservation of tissues from ancient human remains found in a glacier in Canada.

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AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 137:348–355 (2008)
Brief Communication: State of Preservation of
Tissues From Ancient Human Remains Found
in a Glacier in Canada
Maria Victoria Monsalve,1* Elaine Humphrey,2 David C. Walker,1,3 Claudia Cheung,4
Wayne Vogl,5 and Mike Nimmo1
1
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver,
British Columbia V6T 1Z3, Canada
2
Bio-imaging Facility and Microscopy Consultant, Vancouver General Hospital,
University of British Columbia, Vancouver, British Columbia V5Z 1M9, Canada
3
James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia
at St. Paul’s Hospital, Vancouver, British Columbia V6Z 1Y6, Canada
4
Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
5
Cellular and Physiological Sciences, University of British Columbia, Vancouver,
British Columbia V6T 1Z3, Canada
KEY WORDS
cryopreservation; electron and light microscopy; ultrastructure; ancient remains
ABSTRACT
Ancient remains preserved in glaciers
present a unique opportunity for us to advance our
knowledge of human origins, diversity, and health, a
central focus of anthropological studies. Cellular components of hard and soft tissue from frozen human remains
dated between 1670 to 1850 cal AD recovered from a glacier in Canada were studied. Despite the expected ice
crystal damage in some samples, regions of recognizable
structure and ultrastructure were observed. We found
In August 1999, hunters discovered the remains of a
young man frozen in a glacier in the Tatshenshini-Alsek
Park in northern British Columbia (BC), Canada. Initially, carbon dating of his hat and animal skin clothing
indicated an age circa 500 years (Beattie et al., 2000).
Data obtained from the analysis of his robe in combination with direct bone collagen dating suggested that the
human remains are circa 250 years (Richards et al.,
2007). Beattie et al. (2000) described the body as separated at the neck and pelvic areas as a result of postmortem thermal cracking and slumping of the glacier. The
pelvis was discovered protruding from the melting ice
and cervical vertebrae of the trunk were exposed at the
surface of the ice. The skull and several other remains
were found four years later as the glacier receded. This
discovery is officially referred to as Kwäday Dän Ts’inchi
‘‘Long-Ago Person Found’’ by the Champagne and Aishihik First Nations people.
Custody was granted to the local Champagne and
Aishihik people, who allowed approved scientific
research to continue beyond the cremation of the body in
2001. These remains provided a unique opportunity to
assess the state of hard and soft tissue preservation in
frozen remains of this age. The first histological investigation of frozen tissues of this kind was that of a woman
found on St. Lawrence Island, Alaska, radiocarbon-dated
400 AD (Zimmerman and Smith, 1975). Eight mummified bodies carbon-dated 1475 AD (650 years) found in
1972 in Qilakitsoq, northwest of Greenland, have also
been studied (Hart Hansen and Nordqvist, 1996).
Results of extensive pathological studies of two well-preC 2008
V
WILEY-LISS, INC.
that the state of preservation was tissue specific and
that in some tissues the organelles were better preserved
than in others. Skeletal, connective, nervous, and epithelial tissues were recognizable in some of the samples.
DNA had been previously extracted from these remains
and this study illustrates that the ability to successfully
extract DNA may correlate with good preservation of histology. Am J Phys Anthropol 137:348–355, 2008. V 2008
C
Wiley-Liss, Inc.
served frozen bodies found in Utquiaqvik near the most
northern part of Alaska were reported by Zimmerman
and Aufderheide (1984) and Zimmerman (1985). These
two female bodies were radiocarbon dated 1520 AD 6 20
years old. The Tyrolean Ice Man found in the Ötztal
Alps in 1991 is the oldest reported frozen mummy, aged
between 5,100 and 5,300 years old (Hess et al., 1998).
Biochemical techniques allow an estimation of the
state of preservation in ancient remains by quantification of nitrogen and carbon of proteinaceous material.
Data from these techniques are used to predict the suitability of a tissue for DNA analysis. In our previous
study, nitrogen and carbon measurements indicated the
presence of large amounts of proteinaceous material
including collagen in both soft and hard tissues in the
Kwäday Dän Ts’inchi remains (Monsalve et al., 2002;
Grant sponsors: University of British Columbia Faculty of Medicine Summer Student Fellowship Program (2006) and the Summer
Student Research Program (2007).
*Correspondence to: Maria Victoria Monsalve, PhD, UBC Faculty
of Medicine, Life Sciences Centre, Room 1546, 2350 Health Sciences
Mall, Vancouver, BC, Canada V6T 1Z3.
E-mail: vmonsalve@pathology.ubc.ca
Received 27 February 2008; accepted 14 April 2008
DOI 10.1002/ajpa.20864
Published online 7 July 2008 in Wiley InterScience
(www.interscience.wiley.com).
TISSUE PRESERVATION IN ANCIENT FROZEN REMAINS
Monsalve et al., 2003). This encouraged us to proceed
with extraction and amplification of mitochondrial DNA
(mtDNA). In fact, we successfully showed that the
mtDNA of the Kwäday Dän Ts’inchi remains fit in the
context of Native American lineages (Monsalve et al.,
2002; Monsalve and Stone, 2005). Demonstration of
adequate levels of nitrogen and carbon along with successful recovery of mtDNA suggested to us that it might
also be worthwhile to determine the extent of preservation of hard and soft tissue histology. It has been
inferred that histological preservation of bone is a good
predictor of the recovery of DNA from human ancient
remains (Colson et al., 1997). A significant association
between gross and histological preservation and DNA survival was observed in a goose humerus from an ancient
Anglo Saxon site using light microscopy (Haynes et al.,
2002). In this study we employed light and electron microscopic analysis to determine the state of preservation
of the cellular architecture and ultrastructure of both
hard and soft tissues frozen for about 250 years. Furthermore we evaluated the preservation of the hard tissues using the semiquantitative approach of (Hedges et
al., 1995). We discuss the relevance of a histological
analysis of hard and soft tissue structural integrity
to the decision as to whether or not mtDNA analysis is
feasible.
MATERIALS AND METHODS
The protocol and consent form for the study of Kwäday
Dän Ts’inchi remains was approved by the Clinical
Research Board Ethics Committee at the University of
British Columbia. Most of the tissues used in this study
came from the first sampling of the remains being kept
at 2178C at the Royal British Columbia Museum. Standard precautions were taken to minimize contamination
from contemporary sources. We removed hard and soft
tissues from the remains with a sterilized scalpel. Vertebral bone and heart tissues were obtained later on different occasions from the Museum. Lung was obtained subsequently from the University of Saskatchewan where
they were looking for the presence of microorganism
DNA. Our samples were transported under dry ice and
kept at 2808C before processing. Some arm muscle and
vertebral bone samples were conventionally processed
through paraformaldehyde and paraffin sectioning for
light microscopy. Arm, gluteus and thigh muscle, along
with vertebral and humerus bone were processed for
electron microscopy through conventional glutaraldehyde/osmium, and embedded in epoxy resin for thin sectioning. Other frozen lung and heart tissues were freeze
substituted through acetone and osmium for transmission electron microscopy.
Analysis of cellular components
As a preliminary step in the assessment of the state of
preservation, we analyzed hard (compact bone) and soft
(skeletal muscle) tissues with light microscopy. After
formaldehyde fixation, decalcified vertebral bone and
muscle tissue from the left arm were embedded in paraffin, sectioned and stained with Hematoxylin and Eosin
(H & E), Masson’s trichrome and Van Gieson. For categorizing histological preservation, we followed the histological preservation index proposed by Hedges et al.
(1995), as follows: 0, no original feature identifiable,
other than Haversian canals; 1, small areas of well-pre-
349
served bone present, or some lamellar structure preserved by pattern of destructive foci; 2, clear lamellate
structure preserved between destructive foci; 3, clear
preservation of some osteocyte lacunae; 4, only minor
amounts of destructive foci, otherwise generally well preserved; 5, very well preserved, virtually indistinguishable from fresh bone.
Ultrastructural analysis
Small pieces (1–2 mm) of the bicep muscle and bone,
vertebral bone, gluteus, and superficial thigh tissues
were fixed in 2.5% glutaraldehyde either overnight or
using a Pelco 3450 microwave with a coldspot, for
12 min (2 on, 2 off, 2 on, 32) at 258C. The tissues were
washed in sodium cacodylate buffer (0.1 M, pH 7.3–7.4),
and postfixed in osmium in the microwave. The tissues
were dehydrated through a graded series of alcohol and
infiltrated in epon/araldite resin in the microwave and
then polymerized at 608C overnight. Thin sections of
about 70 to 90 nm were cut using a diamond knife and
mounted on 200 mesh formvar coated copper grids. The
sections were stained with 2% uranyl acetate and Reynold’s lead citrate and digitally imaged on a Hitachi H7600 TEM.
For the tissues processed by freeze substitution, we
used a Leica Automatic Freeze Substitution unit. First,
tissues were fixed in acetone with 1% glutaraldehyde
and 0.1% UA at 2858C and then in osmium at 2308C.
Resin infiltration was at room temperature using the
Pelco 3450 Laboratory Microwave. The resin was polymerized at 608C overnight.
RESULTS AND DISCUSSION
Our analyses of soft and hard tissues by light and
electron microscopy in the Kwäday Dän Ts’inchi remains
indicated preservation of recognizable tissues of: 1)
extracellular matrix architecture of soft connective tissues; 2) the architecture of hard skeletal tissue; 3) peripheral nervous tissue; 4) intracellular components of
muscle and epithelial tissues. This data supports previous findings wherein successful DNA retrieval accompanied good preservation of bone histology (Colson et al.,
1997). Quantitative analysis of cell and tissue components from internal organs may provide verification of
the possibility for successful extraction and analysis of
mtDNA without external contamination.
Connective tissue (collagen)
The glacial environment, where the Kwäday Dän
Ts’inchi remains were discovered, helped to preserve the
collagen. Collagen was the best preserved element of the
connective tissue of the skeletal muscle. In fact the collagen retained its affinity for Van Gieson and Masson’s trichrome. Electron microscopically, type I collagen
retained its characteristic banding pattern with a periodicity of 47.5 nm and fibril diameters of 60 nm. We
encountered this level of collagen preservation in the
skeletal muscle, lungs, and heart (Fig. 1A–D).
These observations are consistent with those from
eight frozen mummies found in a shelter in Qilakitsoq,
Greenland (Ammitzbøll et al., 1989; Kobayasi et al.,
1989), dated at 500 years old (Ammitzbøll et al., 1989)
also demonstrating well-preserved collagen fibrils with
American Journal of Physical Anthropology
350
M.V. MONSALVE ET AL.
Fig. 1. Electron micrographs of fibrillar collagen from three different soft tissues. (A) Type I collagen from skeletal muscle of
the biceps in long section. Microwave processed. (B) Type I collagen from skeletal muscle of the biceps in cross section. Microwave
processed. (C) Fibrillar collagen from lung. Processed by freeze substitution. (D) Fibrillar collagen from heart. Processed by freeze
substitution.
the characteristic axial periodicity of 66 nm in one of the
eight mummies as seen with modern human skin samples (Junquiera et al., 1995). By cellulose electrophoresis, they demonstrated the persistence of collagen associAmerican Journal of Physical Anthropology
ated with glycosaminoglycans. Kobayasi et al. (1989)
also demonstrated good preservation of collagen along
with other components of the skin in one of the other
mummies from the same shelter.
TISSUE PRESERVATION IN ANCIENT FROZEN REMAINS
351
Fig. 2. Vertebral bone. (A) Light micrograph of transverse section through a lumbar vertebral bone. Haematoxylin & Eosin
stained. Haversian canals (hc), concentric rings of lamellae (cr), and lacunae (l). (B) Electron micrograph of concentric rings (cr)
and canaliculi (c) of vertebral bone in longitudinal section. Microwave processed. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
Even in a significantly older glacial mummy (the Tyrolean Ice Man dated 5,300 years old), type I collagen was
found to be well preserved in soft tissues (Hess et al.,
1998). In both the Tyrolean Ice Man and the Kwäday Dän
Ts’inchi remains, it was possible to successfully analyse
and establish mtDNA lineages (Handt et al., 1994; Monsalve et al., 2002; Monsalve and Stone, 2005). MtDNA
analysis established the European background of the Tyrolean Ice Man and we successfully established the
Native American aboriginal background of the Kwäday
Dän Ts’inchi man. Analyses by light and electron microscopy may establish the persistence of a cellular environment from which DNA can be extracted. MtDNA analysis
was successful in two of the four frozen corpses that had
ultrastructure recognizable type I collagen (Handt et al.,
1994; Monsalve et al., 2002). Therefore, one might predict
that it would be valuable to perform mtDNA analysis on
the corpses found in Qilakitsoq, Greenland.
Skeletal tissue (compact bone)
Analysis of tissue by light (LM) and electron microscopy (EM) indicated recognizable internal architecture in
the vertebral bone. The Haversian canals and lacunae
were conspicuous in light microscopy (Fig. 2A). A numerical range from 0 to 5 was previously proposed to categorize the extent of the survival of bone structure (Hedges
et al., 1995). The vertebral bone of the Kwäday Dän
Ts’inchi remains fell in category 3 because of the persistence of lamelli and lacunae in the concentric rings, visible in LM (Fig. 2A). Concentric rings and canaliculi are
seen in EM (Fig. 2B). The humerus bone from which the
Fig. 3. Electron micrograph of a myelinated axon of a
peripheral nerve. The single axon (a) with possible cytoskeletal
elements is enveloped in membranes of the myelin sheath (m).
Fibrillar collagen (c) still surrounds the myelinated axon.
Microwave processed.
American Journal of Physical Anthropology
352
M.V. MONSALVE ET AL.
Fig. 4. Muscle (biceps and heart). (A) Light micrograph of biceps brachii muscle in longitudinal section with cross striations (s).
(B) Electron micrograph of heart tissue. Presumed mitochondria (mt) and myofibrils (m). Processed by freeze substitution. [Color
figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
mtDNA was extracted, exhibited between category 2 and
3. The successful recovery of mtDNA from the Kwäday
Dän Ts’inchi remains supports the finding that preservation of the ultrastructure of the bone is a good predictor
of recovery of DNA (Colson et al., 1997; Haynes et al.,
2002) at least for category 2–3. Therefore, we suggest
that perhaps category 2 and 3 by Hedges et al. (1995)
should be combined to constitute a single category. Furthermore, for those remains where soft tissue is present
a new set of criteria of histological preservation should
be pursued for evaluating the likelihood of obtaining
amplifiable DNA. The extraction of DNA in soft tissue of
the Kwäday Dän Ts’inchi further supports the value of
evaluating histologically soft tissue preservation.
Osteons, their Haversian canals, cement lines, and
osteocytic lacunae have been observed and documented in
other studies of ancient remains (Stout and Teitelbaum,
1976; Stout, 1978), and it has been shown that the geometry of bone structure can be preserved essentially intact
even during fossilization (Stout, 1978). As described by
Colson et al. (1997), the separation of category 2 and 3 is
not clear and we also had this difficulty fitting our data to
the ranking criteria proposed by Hedges et al., 1995.
Nervous tissue (peripheral nerves)
A single myelinated axon of a peripheral nerve associated with the biceps brachii muscle was identified in
these remains. (see Fig. 3).
A nerve with perineurium, Schwann cells, and endoneurium has been observed in one of the similar aged
mummies found in the rock shelter in Qilatkisoq, west
American Journal of Physical Anthropology
Greenland (Kobayasi et al., 1989). The recognizable ultrastructural pattern of a peripheral myelin sheath was
also observed in the skin from the older Tyrolean Ice
Man (Hess et al., 1998). However, the latter nerve axon
appeared amorphous. The existence of reasonable preservation of nervous tissues in frozen remains also supports
the possibility for the successful extraction of DNA to establish human origins, diversity, and health in spite of
nervous tissue being particularly prone to necrosis.
Muscle (biceps brachii, gluteus maximus, and
heart) and epithelium (respiratory tract) tissues
Light microscopic observation of hematoxylin and eosin (H & E) stained biceps muscle revealed the presence
of characteristic of skeletal muscle striations (Fig. 4A).
The presence of striations in skeletal muscle has also
been reported in a 21,000-year-old frozen mammoth
(Zimmerman and Tedford, 1976).
State-of-the-art techniques of microwave processing
and freeze substitution for electron microscopy of arm,
gluteus, and heart muscle ensured the least amount of
extraction of material from the tissues. In some of the
arm and gluteal muscle samples, the outlines of cells/
fibers, were recognizable, but their internal architecture
had deteriorated. In heart tissue (Fig. 4B), preservation
was somewhat better with myofibrils and mitochondria
being visible. In the skin of the thigh, there were neither
recognizable epithelial cells in the epidermis nor muscle
fibers in the dermis.
In the 5,300-year-old Tyrolean Ice Man, the internal
architecture of the muscle tissue was generally disinte-
TISSUE PRESERVATION IN ANCIENT FROZEN REMAINS
353
Fig. 5. Electron micrographs of lung tissue. (A) Possible pneumocyte type II with condensed heterochromatin in the nucleus (n).
Rough endoplasmic reticulum (rer), lipid drops (l) and lysosome (ly) are also seen. Processed by freeze substitution. (B) Lysosome
(ly) and lipid drops (l). Processed by freeze substitution. (C) Profiles of rough endoplasmatic reticulum (rer). Processed by freeze
substitution. (D) Mitochondria (mt) and rough endoplasmatic reticulum (rer). Processed by freeze substitution.
grated and therefore no images were provided at the EM
level (Hess et al., 1998). We attribute the poor muscle
ultrastructure in our sample to ice crystal damage and
autolysis.
In contrast to muscle, the freeze substituted lung tissue retained a remarkable amount of internal cellular
architecture in our samples. In well-preserved pneumoctyes, we were able to identify several subcellular compoAmerican Journal of Physical Anthropology
354
M.V. MONSALVE ET AL.
nents including nucleus, rough endoplasmic reticulum
(rER), lipid drops, lysosomes, mitochondria, and cytoskeletal elements (Fig. 5A–D). Anatomical and cellular
structures along with DNA have also been found in an
8,000-year-old human brain recovered from a swamp in
the Windover archaeological site in Florida (Doran et al.,
1986). The findings of extractable DNA and the presence
of anatomical and cellular architecture together in both
the Windover corpse and in the Kwäday Dän Ts’inchi
remains suggest that whenever there are ultrastructurally recognizable details in corpses like these, there
should also be a sufficient amount of suitable DNA for
extraction and amplification. Likewise whenever extractable DNA is present, some cellular architecture may also
be recognizable.
The preservation of cellular components was found to
be tissue specific and likely dependant upon the history
of freezing and thawing in both field and subsequent
processing. For example, recognizable structures occurred in tissues such as the lung in the upper body
that had limited or no history of exposure and thawing
at the time of excavation from the glacier. With the
exception of fat storage granules, ultrastructure of
the gluteus tissue was poorly preserved. The gluteus of
the lower body had been exposed and thawed from the
ice at the time of the excavation. The samples of arm
muscle came from tissues thawed in the laboratory for
DNA extraction prior to fixation. The upper body was
excavated from the ice and kept frozen. Taken together
these observations of preservation variability suggest
that samples for ultrastructural analysis should be confined to tissues that apparently have not thawed and
refrozen if possible.
Excavation of the Kwäday Dän Ts’inchi remains lasted
for about 1 hour. These remains were flown frozen to
Whitehorse and stored at the Yukon Heritage Branch at
2178C (Beattie et al., 2000). Subsequently they were
kept at 217 or 2198C at the Royal British Columbia
Museum in Victoria and only thawed when taking tissue
samples for analysis and conducting medical imaging.
There are indications that some portions of the body had
remained frozen from the time of death. At death the
individual was probably covered with snow and soon
incorporated into the glacier (Al Mackie, personal communication). A return visit to the site a year later
(Beattie et al., 2000) revealed that the location of the discovery had not been further exposed. Possible exposure
of the remains to thawing and freezing during the last
300 years in the Tatshenshini-Alsek glacier seems
unlikely based on our ultrastructural observation.
To minimize or prevent the thawing of samples, we
used the freeze substitution technique in tissue samples
taken from unthawed portions of the remains. For other
samples the tissues were allowed to thaw and were then
fixed in a Pelco laboratory microwave. However, even in
the freeze substitution material, we observed evidence of
two types of crystal formation in the Kwäday Dän Ts’inchi heart muscle. Crystal formation was likely increased
by thawing and refreezing of the sample (Erk et al.,
1999). In another study, repeated freezing and thawing
of the Tyrolean Ice Man was deemed responsible for
rearrangements of myelin constituents (Hess et al.,
1998).
Adipocere has been found in the Tyrolean Ice Man and
other corpses released by glaciers. A corpse found in a
glacier of the Stubai Alps in 1991 presented well-preserved soft tissues at the gross level by the fatty wax
American Journal of Physical Anthropology
type of mummification (Ambach et al., 1992). Extended
transformation of the body by the fatty wax type of
mummification was also observed in a corpse found in a
glacier of the Ötztal Alps in 1992 (Bereuter et al., 1996).
However, a consequence of adipocere formation is almost
complete structural breakdown in any kind of soft tissue
at the histological level obtained from present day glacier corpses. Adipocere formation is an indication of periods of thawing which allow for saponification of body
fats. In gross observation, Beattie et al. (2000) showed
significant adipocere formation on the back and in the
spine of the Kwäday Dän Ts’inchi remains. Although the
tissues we examined microscopically were from an adipocere-free region, we still observed some localized areas of
cell and tissue deterioration.
As in other studies of frozen mummies, we did find
evidence of shrinkage in some cellular structures. In
some structures, such as in elements of the extracellular
matrix (ECM), we found no shrinkage as in the type I
collagen band in skeletal muscle and heart tissue. A
banding periodicity of 64 nm has been seen in freshly
fixed tissues (Junqueira et al., 1995). However in lung
samples the periodicity was measured at approximately
20 nm. A conducting airway from the lungs of a young
adult male contained a type I collagen with a banding
periodicity ranging from 52 nm to 66 nm and a diameter
of 68 nm (Walker, personal communication).
In the remains of late Pleistocene mammals, considerable postmortem decay apparently occurred before they
were entombed beneath the permafrost (Zimmerman
and Tedford, 1976). The replacement of tissue by masses
of bacteria observed by Zimmerman and Tedford are consistent with this observation. Our observations of only a
few microorganisms in some of the tissues suggest that
there was little postmortem decay (Monsalve et al., submitted). This implies a very short duration between
death and freezing. In summary, such a short interval
may also have contributed to our observations of some
well-preserved cells and tissues.
ACKNOWLEDGMENTS
We are grateful to the Champagne and Aishihik First
Nations for making this project possible. We thank the
Kwäday Dän Ts’inchi Committee and in particular AP
Mackie from the British Columbia Archaeology Branch
and JA Cosgrove from the Royal British Columbia Museum for facilitating the execution of this project, D
Straathof from the Royal Columbian Hospital and O
Beattie from the University of Alberta for their constant
support. We acknowledge the contribution of H Dyck, J
O’Kusky, WK Ovalle, W Tetzlaff, and B Taylor from the
University of British Columbia; and for technical assistance to: C Krebs, M Fejtek, I Hao, D Horne, A Kilistoff,
and K Rensing from the University of British Columbia.
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American Journal of Physical Anthropology
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found, preservation, glacier, brief, communication, remains, state, canada, tissue, human, ancient
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