Which is the best route of administration for cell therapy in experimental model of small-for size syndrome in rats ?

PURPOSE: To evaluate which is the best route of administration for cell therapy in experimental rat model of small-for size syndrome. METHODS: A total of 40 rats underwent partial hepatectomy (70%) that induces the small-for-size syndrome and were divided into four groups of route administration: intravenous, intraperitoneal, enteral and tracheal. The small-for-size syndrome model was designed with extended partial hepatectomy (70%). The animals were divided into four groups of routes administration: intravenous (n=10) intravenously through the dorsal vein of the penis; intraperitoneal (n=10) intraperitoneally in the abdominal cavity; enteral (n=10) oroenteral with the placement of a number 4 urethral probe and maintained at third duodenal portion; tracheal (n=10) after tracheal intubation. We track the animals and monitor them for 21 days; during this follow-up we evaluated the result of cell therapy application tracking animals using ultrasound, radiography and PET-scan. Statistical analysis was performed using GraphPad Prism Software®. Differences were considered significant with the p<0.05. Data are presented as the median and variation for continuous variables. Comparisons between groups were made using analysis of the imaging test by the researchers. RESULTS: All four groups underwent partial hepatectomy of 70% liver tissue targeting the same weight of resected liver. Initially the PET-scan tests showed similarity in administered cells by different routes. However, in few days the route of intravenous administration showed to be the most appropriated to lead cells to the liver followed by enteral. The tracheal and peritoneal routes were not as much successful for this goal. CONCLUSION: The intravenous route is the best one to cell therapy in experimental rat model of small-for size-syndrome.


Introduction
The Small-for-Size Syndrome (SSS) is seen more often if the transplanted segment of the donor is <30% liver or <0.8% of body weight of the recipient 1 .The SSS is a complex process resulting primarily from ischemia-reperfusion injury (IRI) and portal hypertension associated with mismatch between the graft and recipient size.In the initial period, the molecular events associated with subsequent apoptosis, necrosis, proliferation and regeneration appeared in specific patterns of protein expression [2][3][4] .
The diagnosis of SSS graft is generally based on persistent hyperbilirubinemia, coagulopathy, massive ascites during subacute phase post-transplant or extensive hepatic resection 2,5 .Living donor liver transplantation can reduce the discrepancy between the number of patients on the waiting list for transplantation and the number of donor organs available.
Stem cells have three properties: self-renewal, proliferation and differentiation.They have the potential to generate large numbers of mature cells continuously throughout life 6,7 .The term refers to cells having ability to generate daughter cells with characteristics similar to the mother cell, as well as differentiate into specialized cells 6,7 .Stem cells are able to differentiate into all cell types and can be used in replacement therapies for various diseases.Embryonic and fetal liver cell lines can be an important tool for cell therapy in patients with liver disease source, as they have a high rate of differentiation into hepatocytes and bile duct cells.Although there are numerous published papers on the administration of stem cells, none of them evaluate the best route of administration.The aim of this study was to evaluate which is the best route of administration for cell therapy in experimental rat model of liver regeneration on small-for size-syndrome.

Methods
The study was designed in accordance with the Guide for the Care and Use of Laboratory Animals of University of São Paulo.This project was approved by the Ethics Committee, for the the Guidelines of Animal Experimentation of the University of São Paulo School of Medicine, São Paulo, SP, Brazil.
Two or three animals from the same treatment group were housed per cage.The animals had free access to tap water and standard food during the entire experiment.Food intake was not measured.

Study design
The animals underwent an extended partial hepatectomy (70%) that induce the small-for-size syndrome and were divided into four groups of routes administration (Figure 1): intravenous (n=10) -intravenously through the dorsal vein of the penis; intraperitoneal (n=10) -intraperitoneally in the abdominal cavity; enteral (n=10) -oroenteral with the placement of a number 4 urethral probe and maintained at third duodenal portion; tracheal (n=10) -after tracheal intubation (Figure 2).We track the animals and monitor them for 21 days; during this follow-up we evaluated the result of cell therapy application tracking animals using ultrasound, radiography and PET-scan.All procedures began with the intraperitoneal anesthesia using 5% ketamine hydrochloride 30 mg/kg (Ketalar ® , Cristália).Animals were kept warm with a 45W, 127V halogen bulb and body temperature was monitored using a rectal digital thermometer (YSI 4000A Precision Thermometer, USA) and maintained between 35°C and 37°C.

Incorporation of Propidium Iodide (PI) and reading sample
The fixed and previously stored samples were centrifuged at 2000 rpm for 5 minutes.The supernatant was discarded, the cells resuspended in 1 ml flow cytometry buffer and centrifuged again.
After centrifugation, the supernatant was discarded and the cells resuspended in PI solution prepared from 5 ml PBS (sodium phosphate buffer pH 7.4) to which were added 100 5μL Triton (0.01% v/v) and 50μL of RNAse (2mg/mL) and 20μL of propidium iodide (5mg/mL).
Then, if the held data acquisition FACSCalibur flow cytometer on 10.000 events for analysis and Win Mdi 2.8 program was used.

Evaluation of routs administration of stem cell in rats
Initially, we performed the analysis in the seventh day after surgery; follow by second analysis in fifteenth day and subsequent twentieth day for establishing and detecting the stem cell by PET-scan, ultrasonography and radiography for each route administration group.

Literature review
A literature review was performed with the Mesh-terms "Liver regeneration"; "Disease models, Animal"; "Stem cells" and "Rats".The electronic search was held of the Medline-PubMed, in English.The search was completed in September 2014.

Statistics
Statistical analysis was performed using GraphPad Prism Software ® .Differences were considered significant with the p<0.05.Data are presented as the median and variation for continuous variables.Comparisons between groups were made using analysis of the imaging test by the researchers.

Results
A total of 40 rats underwent partial hepatectomy that induces the small-for-size syndrome and the four groups of routes administration were evaluated in three moments (seventh, fifteenth and twenty-first day).They were carried out as follows: intravenous group (n=10), intraperitoneal group (n=10), enteral group (n=10) and tracheal group (n=10).
All four groups underwent partial hepatectomy of 70% liver tissue targeting the same weight of resected liver.The routes of administration for cell therapy in experimental rat model of small-for size syndrome for each group were evaluated using ultrasound, radiography and displayed below the PET-scan (Figure 4).During follow-up of twenty-one days, the cells remained alive after administration in all groups showed by PET-scan.Initially it was observed in PET-scan tests a similarity between administered cells by different routes.However, after few days the intravenous route showed to be more appropriated to lead cells to the liver, followed by enteral route.The tracheal and peritoneal routes were not as much efficient for this ultimate goal.
The evaluation by ultrasound and radiography of the rats, in the different groups showed no differences to show the marked stem cell (Figure 5).In the laboratory analysis, we observed an elevation of liver enzymes (AST and ALT), slight change of total and direct bilirubin, and extending the coagulation time.Forty percent of the cases presented incoagulable value in the sample (Table 1).
A literature review was performed with the Mesh-terms: "Liver regeneration"; "Disease models, Animal"; "Stem cells" and "Rats" in order to see the administration route used by the authors in this specific field.The initial review search showed 69 articles in Medline-PubMed with the specific Mesh-terms and we analyzed the methods used in each abstract as showed in Figure 6.

FIGURE 1 -
FIGURE 1 -The study design for four groups of different routes administration in experimental small-for-size syndrome.

FIGURE 2 -
FIGURE 2 -The four groups of different routes of administration in experimental small-for-size syndrome.A. Intravenous; B. Intraperitoneal; C. Enteral; D. Tracheal.

FIGURE 3 -
FIGURE 3 -Surgical technique of experimental small-for-size syndrome in rats.

FIGURE 4 -
FIGURE 4 -PET-scan evaluation of the route of administration for cell therapy in experimental rat model of small-for size-syndrome. A. Intravenous; B. Peritoneal; C. Tracheal; D. Enteral.Note: The animal above is the twenty-first day and down the fifteenth day post administration.

FIGURE 5 -
FIGURE 5 -The ultrasound and radiography evaluation the route of administration for cell therapy in experimental rat model of small-for size syndrome.Note: The animal above is the twenty-first day post administration.

TABLE 1 -
Findings of the laboratory biochemical.Note: The median and variation.AST, aspartate transaminase; ALT, alanine transaminase; TB, total bilirubin; BD, direct bilirubin; GGT, gamma glutamyl transferase; AP, alkaline phosphatase; PT, prothrombin time; INR range.*Forty percent of the cases presented incoagulable value in the sample and INR>5.

FIGURE 6 -
FIGURE 6 -The literature review performed with the Mesh-terms: Liver regeneration; Disease models, Animal; Stem cells and Rats showed 69 articles in Medline-PubMed.