The ability to regenerate the liver in experimental animals with induced cirrhosis under the influence of the spatially modulated erbium laser

Scientific practical journal “Surgery News”

SCIENTIFIC PUBLICATIONS EXPERIMENTAL SURGERY 2015 Mar-Apr; Vol 23 (2): 131-137


I.I.Pikirenia 1, A.N.Zemlyanik 1, V.V.Khomchenko 2
SBE “Belarusian Medical Academy of Post-Graduate Education” 1,
Ltd. “Linline Medical Systems” 2, Minsk,
The Republic of Belarus

p. 131-137 of the original issue

Objectives

To study the possibility of the liver regeneration with induced cirrhosis using micro-ablation method with spatially modulated erbium laser radiation (wavelength of 2,936 μm) in experimental animals.

Methods

In the experiment the liver cirrhosis was modeled in 70 albino outbred rats by means of carbon tetrachloride. Cirrhotically changed left liver lobe was treated by micro-ablation method (erbium laser, wavelength of 2,936 μm, impulse duration of 0,3 ms, packed with SMA (Space Modulated Ablation), the right lobe was a control one. The repeated operations with the treating of the liver have been carried out on the 15th, 30th, 60th days, followed by exclusion from the experiment and histological examination of the liver.

Results

After a single treating of the rats’ liver with spatially modulated erbium laser radiation at microscopy on the first day of exposure the signs of pre-existing liver cirrhosis in the hepatic tissue so as on the depth of exposure (from 5,5 to 6,0 μm) the destruction of some hepatocytes were revealed. Starting from the 30th day the regeneration signs of cirrhotically changed liver were established, including the reduction of the connective tissue amount and signs of the portal hypertension. At the same time no signs of regeneration in the right liver lobe not subjected to laser radiation were revealed. On the 60th day after double liver treating neoangiogenesis, proliferation of the bile ducts, reduction of the number of false lobules and connective tissue structures have been registered compared to the right lobe of the liver, which treating is not performed.

Conclusion

The impact of the micro-ablation method on the cirrhotically changed liver causes active process of regeneration that may be indicated for application Bridge-therapy prior to liver transplantation.
Keywords: liver cirrhosis, high energy radiation, liver regeneration, liver transplantation, connective tissue, cell proliferation, Bridge-therapy

Introduction

A large number of scientific works and experimental studies are devoted to the problem of liver regeneration [1, 2, 3, 4, 5, 6, 7]. This is due to the fact that liver diseases are very prevalent, and the problems of effectively treating these diseases are far from being solved. The growing medical and social significance of chronic liver diseases demands renewed effort to develop treatments for these diseases [8, 9].

Cirrhosis of the liver is a diffuse pathological process that is accompanied by excessive fibrosis and the formation of structurally abnormal regeneration nodes [10, 11, 12, 13]. Clinical manifestations of this disease vary and are determined mainly by etiological factors, the intensity of the pathological process, reduced liver function, as well as the degree of development of portal hypertension. Despite the wide range of medicines available, they are not able to stabilise the process in many patients. The high mortality of patients with cirrhosis is due to developed complications that require very costly treatments [14]. There are many palliative techniques that can extend the life of this category of patients, though there is only one radical treatment: organ transplantation.

Liver transplantation is a high-tech and expensive treatment that is limited by the insufficient number of available donor organs. The first step in preparing for a liver transplantation is being included on a waiting list. The creation of a list of patients awaiting liver transplantation and the management of patients before orthotopic liver transplantation are important aspects of the work of any transplant centre, and they are critical to the successful outcome of the operation. Patients included on the waiting list represent potential recipients. Because of the high technical and material difficulties of the individual medical approach to each case and the lack of donor organs, this type of surgery is not an option for all patients suffering from cirrhosis of the liver [15]. Therefore, it is a matter of pressing concern to find new ways to treat portal haemodynamics in cirrhotic livers in order to prolong the life of these patients before liver transplantation. Besides bypass operations, other ways of treating portal haemodynamics in patients suffering from cirrhosis of the liver include, on the one hand, stimulation of intrahepatic neoangiogenesis and regeneration of liver cells and, on the other, reducing the amount of connective tissue in hepatic portal tracts, thereby reducing the manifestations of portal hypertension. This approach to treating the disease provides a kind of “bridge” (medical bridging therapy) between the time when a patient diagnosed with cirrhosis of the liver is placed on a liver transplantation waiting list and the liver transplant operation.

In parallel with efforts to optimise methods to surgically prevent complications of cirrhosis of the liver, researchers continue to actively seek out a variety of modern methods to stimulate liver regeneration. Even if such methods only partially restore the structure and functioning of the liver, they are useful in order to prolong the life of the patient until the time when a liver transplantation can be performed.

The reparative regeneration of the diseased liver is performed selectively and is aimed at restoring the parenchyma-stroma relationship by increasing the amount of functioning parenchyma (through so-called regenerative hypertrophy). When there is prolonged inflammation of the liver (caused by alcoholic, toxic, viral, or autoimmune hepatitis) and adipose degeneration (due to obesity, diabetes or other metabolic disorders), connective tissue is created at the site of cells damaged by cirrhosis. The liver function is impaired, its structure (architectonics) changes, and cirrhosis develops due to the creation of such tissue. Therefore, another possible type of treatment is intracellular and extracellular resorption of the excess collagen. This will make it possible to support the best parenchyma-stroma relationship, adequate blood supply and the functional capacity of the organ. Violation of the reparative regeneration process is a specific prerequisite for chronic inflammation. In this regard, researchers are particularly interested in the study of the impact of high-energy laser radiation produced by modern medical laser equipment on the cirrhotic liver.

Current studies insufficiently address issues of regenerating hepatocytes in diseased livers using modern laser systems.

In this regard, a comprehensive comparative morphological study is in order. It would conduct an experiment that can produce reliable data about
regeneration processes that take place in the diseased liver, their duration and variability when laser radiation is exposed to liver tissue.

The goal of the present paper is to study the possibility of liver regeneration in experimental animals with induced cirrhosis through the microablative method using spatially modulated radiation with a wavelength of 2936 μm produced by an erbium laser.

Materials and methods

The experiment was carried out using the vivarium at the Belarusian Medical Academy of Postgraduate Education research laboratory.
In light of data from previous studies that explored the possibility of liver regeneration as well as our own positive data about the effects of erbium laser radiation on skin scars, we conducted an experiment on white rats in order to study the possibilities of regenerating cirrhotic liver tissue.

We first performed a preliminary experiment to determine the optimum toxic dose and method of administration (subcutaneous or intragastric) of hepatotropic poison (carbon tetrachloride (CTC), which was selected on the basis of research conducted by T.R. Mirsaev [16]) to 15 randomly bred rats (males and females) in order to induce liver cirrhosis in the animals. The animals were kept in vivarium conditions at a temperature of 18-22° C with free access to water. Throughout the experiment, the animals received specialised varied full rations of food. The animals were kept under quarantine for 30 days before the start of the experiment. The study assessed the animals on the basis of macroscopic (body weight, yellowness of the skin, hair loss, visual assessment of the state of the liver during surgery) and microscopic criteria (histological examination of biopsy material obtained during surgery and at the time of the natural death of the animal). Insofar as animal body weight was one of the most objective indicators of the general condition of the organism, the animals were regularly weighed on electronic scales over the course of the experiment.

In accordance with the results of preliminary testing, in the later experiment liver cirrhosis was induced in the rats through intragastric administration of CTC over the course of 2 months in accordance with the methodology that we worked out (“Method for Simulating Cirrhosis of the Liver” Patent MKI G 09B 23/28, authors A.N. Zemlyanik and I.I. Pikirenia).

After 2 months the formation of the cirrhosis was confirmed by taking an ultrasound using the 12 Mhz fingertip sensor of a BK PRO FOCUS (USA) ultrasound scanner and conducting a morphological study. Upon completion of the administration of the CTC to the animals and once a histological picture was obtained of the cirrhosis of the liver, surgery was performed on a control group of the rats.

A further experimental study was carried out under laboratory conditions on 70 pubescent random bred male white rats weighing 160-250 g.

The animals were injected with sodium thiopental and fixed in the dorsal position. After the operation area was treated in sterile conditions, a quick incision was made along a white line of the abdomen. The liver was secured to the incision wound using sutures. During the surgery the cirrhotic left lobe of the liver was treated using the microablative method as applied by an erbium laser. The right lobe of the liver in each animal was not treated, as it served as a control.
The peritoneum was sutured with catgut, the aponeurosis was sutured with a continuous nonabsorbable material, and the skin was sutured with black silk using individual interrupted stitches.

All animals in the experiment were divided into 3 groups.
Group 1 (30 animals) that was subjected to one surgical operation in accordance with the above procedure. Members of this group were then removed from the experiment and subjected to a histological examination of the liver.
Group 2 (30 animals) was subjected to two surgical operations in accordance with the above procedure. For the second surgical procedure, the animals in Group 2 were divided into 2 subgroups (that were subjected to a second microablative treatment performed on the 15th and 30th days of the experiment).
Group 3 (10 animals) was a control group in which the animals’ cirrhotic livers were not treated using an erbium laser.

The surgeries were performed using the Multiline laser device (produced by LINLINE Medical Systems, Minsk, Belarus), which has several independent laser sources, including erbium, neodymium, ruby, and alexandrite. (It is equipped with 7 interchangeable high-energy laser emitters).

The experiment used an erbium laser equipped with a SMA (space modulated ablation) attachment, which ensures the required spatial distribution of energy in the laser beam (Eurasian Application No. 201200845/26 for the invention “Method for Rehabilitating Biological Tissues and Device for Performing this Procedure,” authors V.V. Khomchenko, D.V. Gorbach, A.V. Sukhodolov). The treatment was performed in scanning mode. We used the following radiation parameters:
- Wavelength – 2936 μm;
- Pulse width – 0.3 μm;
- Average energy density – 2.1 J/cm;
- Pulse repetition frequency – 3 Hz.

This type of radiation when applied to the surface of a treated object produces structured areas of microablation that are 50 μm deep and 50 μm in diameter and that are spaced 50 μm apart from each other (50 × 50 × 50 μm).

All procedures that the laboratory rats were subjected to, including euthanasia, were performed in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, which was adopted in Strasbourg on March 18, 1986, and the Rules for Working with Experimental Animals.

After the left lobe of the liver was treated using the microablative method over the course of 2 months on the 1st, 7th, 15th, 21st, 30th, 52nd, and 60th days of the experiment, the animals were removed from the experiment through an overdose of ether anesthesia followed by decapitation. The monitoring process observed the general condition of the rats. The state of the left and right lobes of the liver was visually assessed by taking photographs and then morphologically examining the images. The treated section of liver resulting from the operation was subjected to a histological examination allowing the formulation of a final diagnosis. The section of liver removed for histological examination was placed in a 10% solution of neutral buffered formalin, and paraffin sections were prepared from which Van Gieson stains were made using Sudan III to identify fat vacuoles, their shape, and preferential localization. A periodic acid Schiff reaction was used to detect glyco-proteins, and the dye MSB (Martius-Scarlett-Blue) was used to stain and identify components of connective tissue.

Light microscopy was used to study the overall picture and evaluate the severity of the major histological features of liver cirrhosis, reflecting the state of degenerative, inflammatory-necrotic, and sclerotic regenerative processes in the liver caused by both the following parenchymal and stromal elements: fatty degeneration of hepatocytes, glycogen content in the cytoplasm of hepatocytes, liver cell necrosis, hypertrophy of nuclei of hepatocytes, proliferation of the bile duct epithelium, inflammatory cell infiltration of tracts and parenchyma, and the number of false lobules.

Results

Signs of existing cirrhosis (fatty degeneration of hepatocytes, necrosis of hepatic cells, hypertrophy of nuclei of hepatocytes, inflammatory cell infiltration of tracts and parenchyma, presence of false lobules) were already discovered on the first day after rats in all groups received one liver treatment using the microablative method as applied by a spatially modulated erbium laser during the microscopy on first day. In addition, the treatment depth was designed to target processes responsible for the local destruction of certain hepatocytes. The treatment depth varied between 5.5 to 6.0 mm (Fig. 1).

The liver of a rat from Group 1 after treatment using the microablative method (first day). Van Gieson’s Stain. Enlarged by a factor of 20.

Fig. 1. The liver of a rat from Group 1 after treatment using the microablative method (first day). Van Gieson’s Stain. Enlarged by a factor of 20.

On day 7 a histological examination of the livers of rats from Groups 1 and 2 confirmed the presence of the main features of cirrhosis of the liver: false lobules, fatty liver disease, inflammatory cell infiltration of tracts and parenchyma, periportal sclerosis, and zonal necrosis.

Microscopies performed on the 15th and 21st days revealed changes in the type of destruction of certain hepatocytes noted above in animals from Group 1. No coagulation and coagulative necrosis, which are characteristic of thermal lesions, were noted.

Starting on the 21st day, signs of regeneration of the cirrhotic liver in animals from both Groups 1 and 2, including reduced amounts of connective tissue, were discovered. Signs of liver regeneration were most pronounced in animals from these groups on the 30th day: A group of sequential cellular reactions was discovered in treated areas. These reactions were evidenced by activated platelets accompanied by degranulation and the formation of aggregates, increased numbers of granule cells, increased numbers of macrophages and fibroblasts followed by their increased functional activity, endothelial cell proliferation, and the formation of a new vascular bed. No such signs of regeneration were discovered in areas on the right lobe of the liver in rats from Group 3 that did not receive any treatment.

On the 52nd day after the first surgical operation animals from Group 1 displayed the signs of regeneration described above, but on the 60th day signs of liver regeneration were less pronounced, which was confirmed by the assessed histological features of regeneration.

The liver of a rat from Group 2 (Subgroup 1) after treatment using the microablative method (15 days after the second treatment): 1 - false lobules of the liver with layers of connective tissue; 2 - treated area, which is rich in blood vessels and proliferating bile capillaries. This area lacks layers of connective tissue (depth of radiation exposure of 50 μm). Haematoxylin and eosin stain. Enlarged by a factor of 20.

Fig. 2. The liver of a rat from Group 2 (Subgroup 1) after treatment using the microablative method (15 days after the second treatment): 1 – false lobules of the liver with layers of connective tissue; 2 – treated area, which is rich in blood vessels and proliferating bile capillaries. This area lacks layers of connective tissue (depth of radiation exposure of 50 μm). Haematoxylin and eosin stain. Enlarged by a factor of 20.

The liver of a rat from Group 2 (Subgroup 2) after treatment using the microablative method (at 30 days): Hematoxylin and eosin stain. Enlarged by a factor of 20.

Fig. 3. The liver of a rat from Group 2 (Subgroup 2) after treatment using the microablative method (at 30 days): Hematoxylin and eosin stain. Enlarged by a factor of 20.

In light of the findings, we decided to support regeneration processes in the liver by subjecting the liver to a second laser treatment operation on the 15th and 30th days after the first surgery (for Subgroups 1 and 2, respectively, of Group 2).

After the second treatment using the microablative method, a histological examination of rats from Group 2 revealed changes that lacked the characteristic signs of thermal damage. Such changes were also the case after the first treatment. Local areas of destruction of certain hepatocytes were observed along the entire depth of the treated area. On day 15 after the second laser treatment, rats from Group 2 (Subgroup 1) showed signs of cirrhotic liver regeneration, including reduced amounts of connective tissue (Fig. 2).

As can be seen in Fig. 3, an increased number of granule cells, macrophages and fibroblasts, as well as a proliferation of endothelial cells with the formation of a new vascular bed was noted at 30 days after the second treatment in the areas of the livers of rats (from Subgroup 2, Group 2) that were exposed to microablative treatment. A decrease in the amount of connective tissue in the cirrhotic liver was observed. There were multiple capillaries (neoangiogenesis), a proliferation of bile ducts, and a reduced number of false lobules and connective tissue structures. No such regeneration characteristics were detected in the right lobe of the liver that was not exposed to laser radiation nor in the untreated control group (Group 3) when histological material was taken and examined at different times (Fig. 4).

The right, untreated control lobe of the liver in rats from Group 2 (Subgroup 2) (60 days). Haematoxylin and eosin stain. Enlarged by a factor of 20.

Fig. 4. The right, untreated control lobe of the liver in rats from Group 2 (Subgroup 2) (60 days). Haematoxylin and eosin stain. Enlarged by a factor of 20.

Thus, signs of regeneration of liver tissue were most pronounced on the 30th day after the second treatment in rats from Subgroup 2, Group 2 as compared to rats from Subgroup 1, Group 2. Rats with cirrhosis of the liver in the control group (Group 3) showed no signs of liver regeneration. In particular, we observed broken lobular structure of the liver, multiple false lobules, fatty degeneration of hepatocytes, hypertrophy of nuclei of hepatocytes, and inflammatory cell infiltration of portal tracts and parenchyma in these rats.

Rats from Group 3 (control) continued to exhibit symptoms of liver cirrhosis, which was confirmed histologically and by ultrasonic diagnostic data, throughout the experiment as well as at the time of its completion. On the 52nd and 60th days histological material was taken from rats in Group 1, which underwent one surgical procedure. There were significantly fewer false lobules and less inflammatory cell infiltration of tracts in the field of view as compared with rats from the control group. On the 52nd and 60th days rats in Subgroup 2, Group 2 exhibited very pronounced signs of regeneration as compared to rats from Group 1 and Subgroup 1, Group 2, namely they demonstrated endothelial cell proliferation with the formation of a new vascular bed (neoangiogenesis).

Discussion

The changes that emerged after a single treatment of the rats’ livers using the microablative method as applied by a spatially modulated erbium laser were determined to be the mechanical destruction of individual cells. No coagulation and coagulative necrosis, which are characteristic of thermal legions, were noted.

In our view, the mechanical destruction of biological tissue occurs as a result of the following processes. Thanks to the special attachment, the laser radiation is able to cause microablation (50 × 50 × 50 μm in size), generating powerful acoustic waves that act against the surface of the treated object. Since the microablation occurs simultaneously at many points, the acoustic waves that are generated by it are in phase, which makes the process of their interference possible. The energy density of the laser pulses was chosen in such a way that after the conversion of the laser energy into acoustic waves, the power of the acoustic waves would not be great enough to independently damage treated biological tissue. However, when neighboring waves interact locally, the force of their impact increases to the extent necessary to ensure the mechanical destruction of tissue located in these areas. Given that the mechanism that impacts the cells is not thermal, and the areas of local exposure can be approximated to the dimensions of individual cells, this type of destruction produces portions of destroyed tissue (cells and their membranes) while leaving surrounding areas undamaged. This is confirmed by data from the microscopy performed on the first day after the liver treatment.

Thus, the impact of using a spatially modulated erbium laser with a wavelength of 2936 μm (microablative method) on a liver with induced cirrhosis in experimental animals spurs regenerative processes in the lobe of the liver that is treated in this manner. The results confirm the known fact that a cirrhotic liver has high regenerative potential. Further research is needed to enhance the effectiveness of the microablative method and to explore the possibility of clinically applying the method to treat the end stages of cirrhosis of the liver.

Conclusions

1. The microablative method has the effect of causing mechanical damage to certain hepatocytes in a cirrhotic liver.
2. The microablative method spurs an active regenerative process in a cirrhotic liver, including neoangiogenesis and the formation of bile ducts.
3. The regenerative process is most pronounced when two microablative treatments are conducted with a 30-day interval between treatments.
4. The microablative method may be a viable treatment for the end stages of cirrhosis. It can be used to spur regeneration processes as a bridge therapy for patients awaiting liver transplantation.
5. Further research is needed in order to explore
which radiation parameters should be adjusted in order to increase the depth of impact and enhance the effect of the regeneration of the cirrhotic liver.

The author has no conflict of interest

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