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Title page
Biological influence of extracts of cryopreserved fragments of piglets’ heart
and skin
A running title
Biological effect of extracts of organs
Liliia A. Rohoza*, Iryna G. Bespalova, Mykola O. Chizh, Sergiy Ye. Halchenko,
Boris P. Sandomirsky
Department of Experimental Cryomedicine, Institute for Problems of Cryobiology
and Cryomedicine of the NAS of Ukraine, 23, Pereyaslavska srt., Kharkiv 61015,
Ukraine
*Author for correspondence (l.rohoza@runbox.com)
L.R., 0000-0001-6095-5923
KEY WORDS: Cold wound, Myocardial ischemia, Peptides, Regeneration
Summary statement
The extracts of cryopreserved fragments of piglets’ heart or skin were shown as
stimulating reparative regeneration of heart tissues in myocardial ischemia of rats
and skin in a cold wound, respectively.
ABSTRACT
One of the new directions on which the searches used to find the methods to
effectively correct the regeneration in case of different pathologies is the
application of biologically active peptides and their mixtures. In the work there
was investigated the biological influence of extracts of cryopreserved fragments of
skin and heart of newborn piglets with the cold wound of skin and myocardial
ischemia in rats respectively. For investigations the extracts were obtained from
cryopreserved fragments of newborn piglets’ skin and heart. Cold wound of skin
was modelled in rats by 10 mm copper applicator cooled in liquid nitrogen down to
-196°
С
; the areas of wounds were determined by planimetric method, the white
blood cells’ counts were analysed. In rats with myocardial ischemia there were
studied the electrocardiograms, heart rate variability and proliferative activity of
heart cells. The injection of extracts of cryopreserved fragments of skin to the
animal’s abdominal cavity accelerates the healing of cold wound of skin and
normalizes the response of immune system to an injury. After the injection during
2 months to the animals with myocardial ischemia with extracts of cryopreserved
fragments of heart the normalization of electrophysiological indices of heart
activity was observed that testified about the improved blood supply to a heart
muscle. Being injected to healthy animals and those with myocardial ischemia the
extracts of cryopreserved fragments of heart resulted in an increase in proliferative
activity of heart cells. The studied extracts have a high biological effect and can be
applied when designing the drugs for regenerative medicine.
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INTRODUCTION
Numerous investigations have convincingly proven that the main systems
(nervous, endocrine, immune) which are responsible for maintaining the
homeostasis and adequate response to various effects, have a common mechanism
of chemical regulation. Underlying this mechanism is the production and secretion
of a number of substances having a peptidic nature. These molecules have
generally as regulatory peptides. Big attention in modern biology and medicine is
paid also to the study of involvement of peptidic nature’s substances in regulation
of homeostasis of some cell populations and to their role as the signal molecules,
providing the communicative relationships in the norm and under pathological
states (Rehfeld and Bundgaard eds, 2010). Physiologically active peptides were
isolated virtually from all the inner organs, they have the ability of regulation of
functional and proliferative activity of cells in the tissues, which are the initial
material for their obtaining. Therefore the studying of their biological activity is
deemed actual and important (Ivanov, 2010; Shpakov, 2013).
The peptidic bioregulators have been suggested to be a tissue specific. So,
every complex of peptides can to specifically affect a physiological state of the
tissues from which it has been obtained via the nonspecific influence to a body as a
whole (Khavinson et al., 2014).
It was shown that the application of cryobiological techniques when
obtaining the extracts from organ fragments of pigs and piglets allows to increase
the yield and biological activity of peptides which containing in them (Rohoza et
al., 2014a). There were matched the data obtained by MALDI-ToF method about
the molecular masses of peptides in the extracts of cryopreserved fragments of
piglets’ skin and heart with the known peptides from the Protein Knowledgebase
(UniProtKB) (Rohoza et al. 2014b).
The research aim was to determine the biological influence of extracts of
cryopreserved fragments of newborn piglets’ skin and heart in the cold wounds of
skin (CWS) and myocardial ischemia (MI) in the rats respectively.
MATERIALS AND METHODS
Bioethics
The experiments were carried-out according to the “General principles of the
experiments in animals”, approved by the 4rd National Congress in Bioethics
(2010, Kiev, Ukraine) and coordinated with the statements of “European
convention on the protection of vertebrate animals used for experimental and other
purposes” (Strasbourg, 1985) as well as agreed by the Committee of bioethics of
the Institute for Problems of Cryobiology an Cryomedicine of the National
Academy of Sciences of Ukraine.
Obtaining the extracts of cryopreserved organ fragments.
Extracts of cryopreserved organ fragments were obtained from the skin and heart
of newborn piglets. Hearts and skin were fragmented into 2-5 mg pieces and
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washed thrice with physiological solution (0.9% NaCl, pH 7.4) in ratio 1:10. To
the fragments there was added dropwise the cryoprotective solution of 20% PEO-
1.500 in ratio 1:1, pre-packed into 20 ml plastic vials and frozen at 1º
С
/min
cooling down to -70 º
С
with the following transfer into liquid nitrogen. Material
was thawed on the water bath at 37-40 º
С
and cryoprotectant was washed out with
physiological solution. For the extracts obtaining the fragments were incubated for
60 min in physiological solution. To remove thermolabile proteins the supernatant
was warmed in water bath for 15 min and paper filtered. Concentration of peptides
was measured spectrophotometrically at wave-length of 280 nm and 260 nm
(Schmid, 2001).
Experimental models.
The tissue-specific influence of extracts of cryopreserved organ fragments was
studied in outbred white male rats. Influence of the extract of cryopreserved
fragments of newborn piglets’ skin (PiSE) investigated in the model of cold
damage of rats’ skin, and influence of the extract of cryopreserved fragments of
newborn piglets’ heart (PiHE) was studied in the rats with spontaneously
myocardial ischemia (MI). Extracts were injected to rats into abdominal cavity
once per day during the whole experiment. The dose of peptide was 500
μ
g per 1
kg of animal’s weight.
Part 1. Cold injury modelling. Cold wounds of skin (CWS) were modeled in rats
of 180-210 g by 10 mm copper applicator cooled in liquid nitrogen down to -
196 º
С
, the exposure was twice by 30 seconds. The animals were divided into 2
equal groups with 8 rats each: animals with the CWS which were injected with the
physiological solution (control), and those with wound and injections with PiSE.
The wound area was determined by digital images using Bio Vision software. To
the 3d, 7th, 14th and 21st days of experiment leukocyte counts on smears stained
with azure II – eosin (Romanowsky-Giemsa) were analyzed. The index of blood
leukocytes shift (IBLS) was counted as the ratio of the amount of eosinophils and
neutrophils to the one of monocytes and lymphocytes.
Part 2. Animals with spontaneously arisen myocardial ischemia. Mature rats with
MI were selected according to the analysis of ECG indices against the background
of spontaneously arisen pathology under vivarium conditions. Age of the animals
before the experiment was from 14 to 18 months. Totally there were examined 327
rats among those 18 rats with ischemia of the heart muscle were selected. For ECG
recording the hardware-software complex “Poly-Spectrum” (Neurosoft, Russia)
was used. The selection criterion was the elevation of ST-segment and increasing
of T-wave amplitude, decreasing of R-wave amplitude in I and aVL leads. For
estimation of rats’ organism regulatory systems according to ECG the analysis of
the heart rate variability (HRV) was used. The group of norm (intact) was
consisted of 18 rats without revealed pathological changes. The experimental
group with ischemia of the heart muscle (10 rats) was injected with PiHE during 2
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months. The control rats with ischemia of the heart muscle (7 animals) were
injected with physiological solution in amount of 0.5 ml per 100 g of weight. Rats’
hearts for immune histological investigations were fixed in 10 % solution of the
buffered neutral formalin (Shandon Fixx, USA) during 24 hrs. After dehydration
the tissues were imbedded into paraffin with polymer additives (Richard-Allan
Scientific, USA). Slices with 5mm thickness were made from the paraffin blocks.
After blocking the nonspecific linking of proteins via the protein block (Diagnostic
Biosystem, USA), primary antibody Ki-67 were applied (clone MIB-1, Code
IR626, TRU FLEX, DAKO, Denmark). Primary antibody was visualized with the
detection system DAKO EnVision FLEX+ (DAKO, Denmark). For visualization
of histological structure the processed immunohistochemical samples of the heart
were additionally stained with hematoxylin Mayer (DAKO, Denmark). To the
sections which were used as a negative control instead the primary antibodies a
buffer for antibodies dilution was used. The total number of Ki-67 positive cells
were accounted 5 fields of vision (objective
х
40, ocular
х
10).
Statistical analysis.
The results were processed by the nonparametric method MANOVA with software
SPSS Statistics 17.0. Data were expressed as mean ± the standard error of the
mean.
RESULTS
Part 1. Healing of cold wounds, planimetry. Performed investigations for
determining the healing rate of CWS in rats which were injected with PiSE and
without injections shows that to the 3d day of experiment no statistically
significant differences in the wound area in the control and experimental group
were observed (Table 1). To the 7th day the wound area in the animals with PiSE
injections was in 1.5 times less than in the control animals. As well to the 14th day
it was also 3.8 times less than in the control. Thus beginning from the 7th day the
wound area in the animals which were injected with PiSE was statistically
significantly less than in the control rats. Thereby the studied extracts normalize
the healing of cold wounds in the experiment.
Index of blood leukocytes shift (IBLS).
The level of the IBLS increasing under pathological states shows about activity of
inflammation and disorder in immunological reactivity. To the 3d day after the
CWS modeling the IBLS was considerably higher the norm in both groups of
animals (Table 2). The most expressed differences of IBLS were to the 7th day of
experiment. In the control group rats there was observed its further increasing from
1.01 up to 1.49, in the animals which were injected with the extract there was the
IBLS decrease in contrast to the 3rd day. Following the PiSE injections to animals
with the cold injury the IBLS value made 46% versus the control level. On the
14th day this index been decreased in the all groups of animals. To the 21st day in
the animals injected with PiSE this index returned to the norm. This may testify
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that the injections of extracts reduces the inflammation manifestation and
normalizes an immune response.
Part 2. Analysis of heart rate variability.
In the rats with MI injected with physiological solution during 2 months the HR,
SDNN and CV practically do not change (Table 3). Nevertheless there was
observed decreasing of the LF/HF ratio from 0.4 down to 0.3. The minimal and
maximal recorded duration of cardiointervals in rats was to beginning of the
experiment 100 and 493 ms and at the end of experiment it was 113 and 472 ms,
respectively. In the group of animals with MI after PiHE injections during 2
months a recovery of R wave amplitude was recorded. Elevation of ST segment
has been changed by appearance of a domed T wave that testifies about
normalization of blood supply to the heart muscle. In this group of animals among
all of the studied indices of HRV only the HR was not statistically and
significantly differ from the HR of healthy animals; average HR increased from
381 up to 498 bits per min. To the experiment beginning the minimal recorded
cardiointerval was 127 ms and at the end it was 95 ms. The maximal cardiointerval
was 497 and 148 ms respectively. In the norm these indices were 108 and 144
milliseconds. The clearly expressed normalization of the rest of the indices was
observed. Thus, if to the experiment beginning the SDNN value was higher the
norm in 5.6 times so at the end it was only 1.5 times higher, and its value
decreased in 6.7 times. The initial coefficient of variation exceeds the norm in 12.4
times and after the PiHE injections during 2 months this rise made just 1.8 times.
The LF/HF ratio increased from 0.4 up to 2.8 versus the norm rate of 5.6. After 2
months of injections with the extracts the SDNN, LF and HF values did not
statistically and significantly differ from these indices in the norm. Thus the
analysis of HRV revealed that in the animals with the heart muscle ischemia there
is observed significant decrease in the LF/HF ratio and, hereby, also the preference
of parasymphatetic influences upon the heart. PiHE injection to animals with such
a pathology contributes to normalizing all the examined in this research indices.
Proliferative activity.
In intact animals with no detected pathology of the heart the number of Ki-67
positive cells was 7.9% of the total amount and in the animals with MI – 12.0%
(Table 4). Injections with physiological solution both to intact animals and those
with heart pathologies did not influence the number of these cells. In healthy
animals injected with PiHE the number of Ki-67 positive cells in myocardium after
2 months of the injections was 6.1%. In the rats with MI at this term of observation
the number of these cells was (24.6%).
Thereby, the injection with PiHE increases proliferative activity of cells in
the myocardium in the same way it stimulates the reparative regeneration of heart
which is physiologically inherent to heart muscle.
Thereby the PiSE injection into animal’s abdominal cavity accelerates the healing
of CWS and normalizes an immune response in trauma. After PiHE injections
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during 2 months to the animals with MI there were observed an increase in HR,
recovery of R wave amplitude in ECG, elevation of ST segment was changed by
appearance of a domed T wave testifying to normalization of blood supply to the
heart muscle. The coefficient of variation of heart rhythm decreased in 6.7 times,
and the ratio of LF/HF increased from 0.4 up to 2.8. Herewith there was observed
an increase in the index of the spectrum power of neurohumoral regulation for all
the frequency bands up to the normal level and restoration in the balance of
contributions of sympathetic and parasympathetic divisions of autonomic nervous
system into the heart rate regulation.
The PiHE injection to healthy animals and those with MI led to increasing
the proliferative activity of myocardium cells.
DISCUSSION
Studying the mechanisms of biological influence of the tissue-specific peptides is
an actual task of current molecular biology, physiology and medicine. The main
functions of these peptides are the control of proliferation, differentiation and
elimination of cells from a relevant tissue. It is specific, that their effect herewith is
presented not by the individual components, but by large number of peptides with
variously directed activities. The methods traditionally applied in combined
treatment of wounds (antibiotics, sorbents and others) and various physical
influences to the wound not always allow a sufficient normalization of healing. For
this reason recently there have been re-considered the approaches to wound
treatment (Sood et al., 2014; Murphy and Evans, 2012; Sarabahi, 2012). One of
the novel directions when searching the methods for effective correction of
reparation is the use of different means for regulation of wound healing. For
wounds treatment there are used biologically active substances, such as cytokines,
hormones, neuropeptides, regulatory peptides and synthetic peptides (Evans et al.,
2013; Mu et al., 2014; Borena et al., 2015; Tadokoro et al., 2015; You and
Han, 2014).
Application of the abovementioned active agents enables to balance the
inflammation and regeneration whereat the wound healing occurs and also to
control the course and change of the different stages of these processes. However,
when using the physiologically active medications of mono-component peptides
the impact only on a certain link of the regeneration process occurs. Instead, the
poly-component mixtures containing a wide range of regulatory peptides may
influence several key links of this process simultaneously (or sequentially).
Especially this is important under conditions of an infected wound with strong
bacterial contamination, pronounced disorders of metabolism, microcirculation and
lymphodynamic under which there is marked a discoordination in the system of
autoregulation of wound process. It is noted that thereat the spectra of cytokines
secretion are changing. There are dominated the inhibitory rather than inductive
factors, an insufficient inflow of systemic regulatory peptides to wound is
observed (Schreml et al., 2016; Eming et. al., 2007). Such a damage promotes a
prolonged flowing for all the wound phases, lasting healing of wounds and
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formation of keloid scars. Namely the necessity to eliminate the indicated disorders
in the system of peptidic regulation of wound healing enables to pathogenetically
substantiate the application in a combined treatment of wounds the medications of
immunobiological effect. In recent decades an interest to applying the regulatory
peptides in cardiology has been considerably increased too. In particular, such a
problem as stimulation of reparative regeneration of myocardium under the
ischemic heart disease (IHD) is an acute one and constantly updated by the new
approaches of its resolving. Currently there are extensively investigated the
effectiveness and mechanisms of action involving the cell therapy, growth factors
and various peptides as well (Segers and Lee, 2008; Williams and Hare, 2011;
Yla-Herttuala et al., 2007; Hayek and Nemer, 2011). These studies require not
only advanced experimental and clinical investigations, but also general
conceptions as for the mechanisms affecting regeneration.
Traditional treatment and secondary preventive measures of IHD represent a single
set of measures, including therapy with medication, in particular application of
antianginal, and if necessary also antiarrhythmic drugs, cardiac glycosides etc.
(Ahmed et al., 2008; Dobrev and Nattel, 2010; Shavelle, 2007; Dai and Ge,
2012). The necessity of long-term using of these drugs makes the doctors fairly
uneasy about their application safety, because according to the pharmacological
properties of drugs in patients may appear the side effects. Conducting an adequate
antithrombotic therapy may provoke bleeding, particularly in patients with existing
lesions of gastrointestinal and with the presence of other risk factors. Also might
be increasing the activity of transaminases (Calderon et al., 2010). This once
again proves a need of searching a new alternative and even more effective
treatment methods of IHD which enable not only overcome the problem, but also
avoid some of side effects.
Number of papers already investigated the effect how the mono-, as the
polycomponent peptidic drugs under various pathological states of the heart in vivo
and in vitro. Under effect of an exogenous peptide apelin-12 there were observed
the functional recovery of post-ischemic heart and preservation of cell membranes,
resulted from an aerobic metabolism improvement and anticoagulant protection of
myocardium (Pisarenko et al., 2012). When using the peptide apelin-13 in the
models of myocardial infarction and damages under ischemia/reperfusion of the
heart in animals its cardioprotective effect was shown (Azizi et al., 2013). As well
it has been shown that when injecting into a body the mixtures of peptides under
hypercoagulation conditions there was observed a stimulation of functions of
anticoagulant system and fibrinolytic activity (Grigorjeva and Pyapina, 2010)
etc. These polycomponent mixtures are more perspective and have been
intensively studied.
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List of symbols and abbreviations
CV coefficient of variation
CWS cold wound of skin
ECG electrocardiograms
HF high frequency component of HRV
HR heart rate
HRV heart rate variability
IBLS index of blood leukocytes shift
IHD ischemic heart disease
LF low frequency component of HRV
MALDI-ToF time-of-flight matrix-assisted laser desorption / ionization
MI myocardial ischemia
PEO polyethylene oxide
PiHE extracts of cryopreserved fragments of heart of newborn piglets
PiSE extracts of cryopreserved fragments of skin of newborn piglets
SDNN standard deviation of the average duration of RR-intervals (NN-intervals)
Acknowledgements
The authors express their gratitude to the Head of Animal Hosue Facility at the
Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine
Lyudmyla Batsunova for her assistance in handling with animals. We are also
thankful to Dr. Kateryna McDonald, School of Health, University of Central
Lancashire, UK for productive discussion and her valuable help in preparing this
manuscript.
Competing interests
The authors have no conflicts of interest and financial obligations.
Author contributions
B.S. – interpretation of the findings, supervision of the experiment design; S.H. –
planning the experiments, deriving the extracts, analysis of the obtained results;
M.Ch. – participation in the experiments on influence of the PiHE injections to the
rats with MI on electrophysiological indices of heart activity; L.R. – investigation
of the effect of PiHE injection on proliferative activity of rats’ heart cells and
PiHE injections to the animals with MI on electrophysiological indices of heart
activity; I.B. – providing the experiments on the influence of the PiSE injections on
the cold wound healing.
Funding
Funding was provided by the National Academy of Sciences of Ukraine on the
topic registered as “Destructive and renewing processes in the tissues in vivo after
effect of biologically active components» to be performed at the Department of
Experimental Cryomedicine”.
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9
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11
Addition
Table 1. Cold wounds’ area (cm2) depending on experimental conditions
Time of
observation, day
Experimental conditions
Control
N=8
Injections of PiSE
N=8
3 4.5±0.4 3.7±0.4
7 3.4±0.3 2.3±0.2*
14 2.3±0.3 0.6±0.1*
21
1
.
5±0
.
1
Healing
Note. * – differences are statistically significant compared to the control,
р
< 0.05
Table 2. Blood leucocytes shift depending on experimental conditions and
observation time
Experimental
conditions Observation time, days
3 7 14 3
Norm
0.21
Control 1.01 1.49 0.70 0.29
Cold wound +
PiSE 0.89 0.69 0.41 0.23
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Table 3. Indices of the heart rate variability in rats with myocardial ischemia
Indices
Animal groups
Injections with saline
N=6 Injections with PiHE
N=6 Norm
N=24
Initial
indices After 2
months Initial
indices After 2
months
HR,
beats per
min
394±31
407±35
381±29
498±371,2
523±45
Minimum
duration of
RR
interval, ms
100 113 127 95 108
Maximum
duration of
RR
interval, ms
493 472 479 148 141
SDNN 53.4±4.4 50.2±4.6 50.8±5.1 13.4±1.1
1,2
9.9±0.6
CV, %
24
.
1±2
.
0
21
.
2±1
.
9
26
.
1±2
.
6
3
.
8±0
.
2
1
2
.
6±0
.
2
LF, % 28.6±2.5 25.5±2.1 27.8±2.9 74.8±7.1
1,2
79.1±6.4
HF, % 72.7±5.4 75.9±6.6 73.3±7.9 26.1±2.2
1,2
21.2±1.8
Note. 1 – differences are statistically significant if compared to the initial indices,
р
< 0.05;
2 – differences are not statistically significant if compared with norm,
р
> 0.05
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not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
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Table 4.
Total quantity and quantity of Ki-67 positive cells in myocardium of rats
Experimental
conditions Time of observation
Initial indices
N=6 After 2 months
N=6
All cells Ki-67
positive
cells
All cells Ki-67
positive cells
Norm
645±42
51±4 – –
Norm+
saline 584±51 43±4
Norm+
PiHE 627±59 78±3
1,2
Myocardial
ischemia+
saline
591±36 71±6
1
532±44 77±5
1
Myocardial
ischemia +
PiHE
601±52
148±9
1,2
Notes. 1 – differences are statistically significant compared to norm,
р
< 0.05;
2 – differences are statistically significant compared to initial indices,
р
< 0.05
.CC-BY-NC 4.0 International licenseunder a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available
The copyright holder for this preprint (which wasthis version posted September 5, 2017. ; https://doi.org/10.1101/184622doi: bioRxiv preprint
