Activation of p53 After Irradiation Impairs the Regenerative Capacity of the Mouse Liver.

Radiation therapy is one of the treatment methods for hepatocellular carcinoma. However, radiation tolerance of the liver is low, and the detailed effect of radiation on liver regeneration has not been clarified. C57BL/6J mice or hepatocyte-specific p53 knockout (KO) mice (albumin [Alb]-Cre Trp53flox/flox ) were irradiated with a single fraction of 10 Gy localized to the upper abdomen. We performed 70% partial hepatectomy (PHx) 24 hours after irradiation. Liver regeneration was assessed by proliferation cell nuclear antigen (PCNA)- and Ki-67-positive hepatocyte ratios and liver-to-body weight ratio after PHx. To establish a fibrosis model, CCl4 was orally administered for 8 weeks. The murine hepatocyte cell line BNL CL.2 (CL2) was irradiated with 10 Gy. Irradiation activated p53, induced downstream p21 in the liver, and delayed liver regeneration after PHx. While PHx increased hepatocyte growth factor (HGF) levels and activated Met with or without irradiation in the regenerative liver, it activated Akt and extracellular kinase 1 and 2 (Erk 1/2) less in irradiated mice than in nonirradiated mice. In CL2 cells cultured with HGF, irradiation suppressed cell growth by decreasing phosphorylated Akt and Erk 1/2 levels, which was abolished by small interfering RNA-mediated p53 knockdown but not by p21 knockdown. Hepatocyte-specific knockout of p53 in mice abolished the irradiation-induced suppression of both liver regeneration and Akt and Erk 1/2 activation after PHx. In the fibrotic mouse model, the survival rate after PHx of irradiated p53 KO mice was higher than that of wild-type mice. Conclusion: p53 but not p21 is involved in the impaired regenerative ability of the irradiated liver.

Upregulation of PDGF Mediates Robust Liver Regeneration after Nanosecond Pulsed Electric Field Ablation by Promoting the HGF/c-Met Pathway.

Nanosecond pulsed electric field (nsPEF) has emerged as a promising tool for hepatocellular carcinoma ablation recently. However, little is known about how nsPEF affects liver regeneration while being applied to eliminate liver lesions. Besides, the impact of nsPEF ablation on liver function should also be taken into consideration in the process. In this paper, we study the impact of nsPEF ablation on liver function by the measurement of serum levels of AST and ALT as well as liver regeneration and relevant molecular mechanisms in vivo. We found that mouse liver function exhibited a temporary injury without weight loss after ablation. In addition, local hepatic nsPEF ablation promoted significant proliferation of hepatocytes of the whole liver with an increase in HGF level. Moreover, the proliferation of hepatocytes was dramatically inhibited by the inhibitor of c-Met. Of interest, the periablational area is characterized by high level of PDGF and a large amount of activated hepatic stellate cells. Furthermore, neutralizing PDGF was able to significantly inhibit liver regeneration, the increased HGF level, and the accumulation of activated HSCs. Our findings demonstrated that nsPEF not only was a safe ablation approach but also could stimulate the regeneration of the whole liver through the activation of the HGF/c-Met pathway by upregulation of PDGF within the periablational zone.

FUNCTIONAL AND CELLULAR EVALUATION OF THE LIVER AFTER LOW-POWER LASER STIMULATION DURING SURGERY.

BACKGROUND: Partial hepatectomy is a surgical intervention of the liver that can trigger its regenerative process, where the residual lobes deflagrate a compensatory hyperplasia, causing its restoration almost to the original volume. Nevertheless, depending on the extent of liver damage its regeneration might be impaired. The low-power laser has been studied with beneficial results. AIM: To investigate the possible functional and mutagenic damage arising from the use of low-power laser used in liver regeneration after partial hepatectomy. METHODS: Fifteen male adult Wistar rats were hepatectomizated in 70% and laser irradiated or not with dose of 70 J/cm2, 650 nm, 100 mW, directly on the remaining liver, during the perioperative period. These animals were divided into four groups: G1 (control, 7 days); G2 (laser, 7 days); G3 (control, 14 days); G4 (laser, 14 days). Were analyzed the liver weight; number of hepatocytes; deposition of collagen fibers; liver function tests: serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, bilirubin and micronucleus test in peripheral blood erythrocyte. RESULTS: The liver weight was greater in G3 and G4 (p=0.001 and p=0.002) compared to other groups. The deposition of collagen fibers in G1 was statistically higher than the other groups (p=0.01). In tests of liver function and micronucleus test was not found significant differences between the studied groups. CONCLUSION: Low-power laser stimulation did not cause loss of liver function or mutagenic damage.

Functional and cellular evaluation of the liver after low-power laser stimulation during surgery / Avaliação celular e funcional do fígado estimulada por laser de baixa potência no transoperatório

ABSTRACT Background: Partial hepatectomy is a surgical intervention of the liver that can trigger its regenerative process, where the residual lobes deflagrate a compensatory hyperplasia, causing its restoration almost to the original volume. Nevertheless, depending on the extent of liver damage its regeneration might be impaired. The low-power laser has been studied with beneficial results. Aim: To investigate the possible functional and mutagenic damage arising from the use of low-power laser used in liver regeneration after partial hepatectomy. Methods: Fifteen male adult Wistar rats were hepatectomizated in 70% and laser irradiated or not with dose of 70 J/cm2, 650 nm, 100 mW, directly on the remaining liver, during the perioperative period. These animals were divided into four groups: G1 (control, 7 days); G2 (laser, 7 days); G3 (control, 14 days); G4 (laser, 14 days). Were analyzed the liver weight; number of hepatocytes; deposition of collagen fibers; liver function tests: serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, bilirubin and micronucleus test in peripheral blood erythrocyte. Results: The liver weight was greater in G3 and G4 (p=0.001 and p=0.002) compared to other groups. The deposition of collagen fibers in G1 was statistically higher than the other groups (p=0.01). In tests of liver function and micronucleus test was not found significant differences between the studied groups. Conclusion: Low-power laser stimulation did not cause loss of liver function or mutagenic damage.

RESUMO Racional: A hepatectomia parcial é intervenção cirúrgica que pode desencadear processo regenerativo, onde os lobos residuais deflagram resposta de hiperplasia compensatória, ocasionando restauração próxima ao seu volume original. Contudo, dependendo da extensão das lesões hepáticas a regeneração pode ser prejudicada. O laser de baixa potência tem sido pesquisado com resultados benéficos no processo de regeneração hepática. Objetivo: Investigar os possíveis danos funcionais e mutagênicos decorrentes da utilização do laser de baixa potência utilizado na regeneração hepática após hepatectomia parcial. Métodos: Quinze ratos adultos Wistar foram hepatectomizados a 70%, irradiados ou não com laser, dose de 70 J/cm2, 650 nm,100 mW, de forma direta sobre o fígado remanescente, durante o período transoperatório. Os animais foram distribuídos em quatro grupos: G1 (controle, 7 dias); G2 (laser, 7 dias); G3 (controle 14 dias); G4 (laser,14 dias). Foram analisados o peso do fígado; número de hepatócitos; deposição de fibras colágenas; teste de função hepática: alanina aminotransferase, aspartato aminotransferase, fosfatase alcalina, gama glutamiltransferase, bilirrubinas e teste de micronúcleo em eritrócitos. Resultados: O peso do fígado apresentou-se aumentado nos grupos G3 e G4 (p=0,001 e p=0,002) comparados aos demais grupos. A deposição das fibras colágenas no G1 foi estatisticamente maior em relação aos demais grupos (p=0,01). Nos testes de função hepática e teste de micronúcleo não foram encontradas diferenças significativas entre os grupos. Conclusão: O laser de baixa potência não ocasionou perda de função hepática ou dano mutagênico.

Mouse CD11b+Kupffer Cells Recruited from Bone Marrow Accelerate Liver Regeneration after Partial Hepatectomy.

TNF and Fas/FasL are vital components, not only in hepatocyte injury, but are also required for hepatocyte regeneration. Liver F4/80+Kupffer cells are classified into two subsets; resident radio-resistant CD68+cells with phagocytic and bactericidal activity, and recruited radio-sensitive CD11b+cells with cytokine-producing capacity. The aim of this study was to investigate the role of these Kupffer cells in the liver regeneration after partial hepatectomy (PHx) in mice. The proportion of Kupffer cell subsets in the remnant liver was examined in C57BL/6 mice by flow cytometry after PHx. To examine the role of CD11b+Kupffer cells/Mφ, mice were depleted of these cells before PHx by non-lethal 5 Gy irradiation with or without bone marrow transplantation (BMT) or the injection of a CCR2 (MCP-1 receptor) antagonist, and liver regeneration was evaluated. Although the proportion of CD68+Kupffer cells did not significantly change after PHx, the proportion of CD11b+Kupffer cells/Mφ and their FasL expression was greatly increased at three days after PHx, when the hepatocytes vigorously proliferate. Serum TNF and MCP-1 levels peaked one day after PHx. Irradiation eliminated the CD11b+Kupffer cells/Mφ for approximately two weeks in the liver, while CD68+Kupffer cells, NK cells and NKT cells remained, and hepatocyte regeneration was retarded. However, BMT partially restored CD11b+Kupffer cells/Mφ and recovered the liver regeneration. Furthermore, CCR2 antagonist treatment decreased the CD11b+Kupffer cells/Mφ and significantly inhibited liver regeneration. The CD11b+Kupffer cells/Mφ recruited from bone marrow by the MCP-1 produced by CD68+Kupffer cells play a pivotal role in liver regeneration via the TNF/FasL/Fas pathway after PHx.

Different doses of partial liver irradiation promotes hepatic regeneration in rat.

The aim of this study is to investigate whether partial liver irradiation promotes hepatic regeneration in rat. Left-half liver of rat was irradiated to 10 Gy, and the Right-half to 0, 5, 10 and 15 Gy, respectively. Then, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels were evaluated on 0 day, 15-day, 30-day, 45-day and 60-day after liver irradiation. Next, the serum HGF, NF-κB and TGF-ß1 levels were also analyzed on 60-day after liver irradiation. Lastly, the cyclinD1 protein expression was appraised by western blots on 60-day after liver irradiation. ALT, AST and ALP levels were reduced compared with that of controls. The serum HGF, NF-κB and TGF-ß1 levels, and the cyclinD1 protein expression in liver irradiation group were increased compared with that of controls group. However, hepatic regeneration of higher dose-irradiated cirrhotic liver was triggered a more enhanced regeneration, compared with that of higher doses group. In summary, these results suggest that different doses of partial liver irradiation promotes hepatic regeneration in rat.

Low-power laser irradiation fails to improve liver regeneration in elderly rats at 48 h after 70 % resection.

The liver regeneration is an important clinical issue after major hepatectomies. Unfortunately, many organs (including the liver) exhibit age-related impairments regarding their regenerative capacity. Recent studies found that low-power laser irradiation (LPLI) has a stimulatory effect on the liver regeneration process. However, its effects in elderly remain unknown. Thus, this study aimed to investigate the main molecular mechanisms involved in liver regeneration of partially hepatectomized elderly rats exposed to LPLI. The effects of 15 min of LPLI (wavelength of 632.8 nm; fluence of 0.97 J/cm(2); total energy delivered of 3.6 J) were evaluated in hepatectomized elderly Wistar male rats. Afterwards, through immunoblotting approaches, the protein expression and phosphorylation levels of hepatocyte growth factor (HGF), Met, Akt and Erk 1/2 signaling pathways as well as the proliferating cell nuclear antigen (PCNA) were investigated. It was observed that LPLI was not able to improve liver regeneration in elderly rats as evidenced by the lack of improvement of HGF and PCNA protein expression or phosphorylation levels of Met, Akt and Erk 1/2 in the remnant livers. In sum, this study demonstrated that the main molecular pathway, i.e. HGF/Met → Akt and Erk 1/2 → PCNA, involved in the hepatic regeneration process was not improved by LPLI in elderly hepatectomized rats, which in turn rules out LPLI as an adjuvant therapy, as observed in this protocol of liver regeneration evaluation (i.e. at 48 h after 70 % resection), in elderly.

Liver regeneration following partial hepatectomy is improved by enhancing the HGF/Met axis and Akt and Erk pathways after low-power laser irradiation in rats.

A simple, easy, and safe procedure aiming to improve liver regeneration could be of great clinical benefit in critical situations such as major hepatectomy, trauma, or hemorrhage. Low-power laser irradiation (LPLI) has come into a wide range of use in clinical practice by inducing regeneration in healthy and injured tissues. However, the effect of LPLI on the process of liver regeneration, especially those related to the molecular mechanisms, is not fully understood. Thus, the aim of the present study was to investigate the main molecular mechanisms involved in liver regeneration of partially hepatectomized rats exposed to LPLI. We used Wistar male rats, which had their remaining liver irradiated or not with LPLI (wavelength of 632.8 nm and fluence of 65 mW/cm(2)) for 15 min after a 70% hepatectomy. We subsequently investigated hepatocyte growth factor (HGF), Met, Akt, and Erk 1/2 signaling pathways through protein expression and phosphorylation analyses along with cell proliferation (proliferating cell nuclear antigen (PCNA) and Ki-67) using immunoblotting and histological studies. Our results show that LPLI can improve liver regeneration as shown by increased HGF protein expression and the phosphorylation levels of Met, Akt, and Erk 1/2 accompanied by higher levels of the PCNA and Ki-67 protein in the remnant livers. In summary, our results suggest that LPLI may play a clinical role as a simple, fast, and easy-to-perform strategy in order to enhance the liver regenerative capacity of a small liver remnant after hepatectomy.

Single liver lobe repopulation with wildtype hepatocytes using regional hepatic irradiation cures jaundice in Gunn rats.

BACKGROUND AND AIMS: Preparative hepatic irradiation (HIR), together with mitotic stimulation of hepatocytes, permits extensive hepatic repopulation by transplanted hepatocytes in rats and mice. However, whole liver HIR is associated with radiation-induced liver disease (RILD), which limits its potential therapeutic application. In clinical experience, restricting HIR to a fraction of the liver reduces the susceptibility to RILD. Here we test the hypothesis that repopulation of selected liver lobes by regional HIR should be sufficient to correct some inherited metabolic disorders. METHODS: Hepatocytes (10(7)) isolated from wildtype F344 rats or Wistar-RHA rats were engrafted into the livers of congeneic dipeptidylpeptidase IV deficient (DPPIV(-)) rats or uridinediphosphoglucuronateglucuronosyltransferase-1A1-deficient jaundiced Gunn rats respectively by intrasplenic injection 24 hr after HIR (50 Gy) targeted to the median lobe, or median plus left liver lobes. An adenovector expressing hepatocyte growth factor (10(11) particles) was injected intravenously 24 hr after transplantation. RESULTS: Three months after hepatocyte transplantation in DPPIV(-) rats, 30-60% of the recipient hepatocytes were replaced by donor cells in the irradiated lobe, but not in the nonirradiated lobes. In Gunn rats receiving median lobe HIR, serum bilirubin declined from pretreatment levels of 5.17 ± 0.78 mg/dl to 0.96 ± 0.30 mg/dl in 8 weeks and remained at this level throughout the 16 week observation period. A similar effect was observed in the group, receiving median plus left lobe irradiation. CONCLUSIONS: As little as 20% repopulation of 30% of the liver volume was sufficient to correct hyperbilirubinemia in Gunn rats, highlighting the potential of regiospecific HIR in hepatocyte transplantation-based therapy of inherited metabolic liver diseases.

Granulocyte colony-stimulating factor and interleukin-1ß are important cytokines in repair of the cirrhotic liver after bone marrow cell infusion: comparison of humans and model mice.

We previously described the effectiveness of autologous bone marrow cell infusion (ABMi) therapy for patients with liver cirrhosis (LC). We analyzed chronological changes in 19 serum cytokines as well as levels of specific cytokines in patients after ABMi therapy and in a mouse model of cirrhosis generated using green fluorescent protein (GFP)/carbon tetrachloride (CCl4). We measured expression profiles of cytokines in serum samples collected from 13 patients before and at 1 day and 1 week after ABMi. Child-Pugh scores significantly improved in all of these patients. To analyze the meaning of early cytokine change, we infused GFP-positive bone marrow cells (BMCs) into mice with CCl4-induced LC and obtained serum and tissue samples at 1 day and as well as at 1, 2, 3, and 4 weeks later. We compared chronological changes in serum cytokine expression in humans and in the model mice at 1 day and 1 week after BMC infusion. Among 19 cytokine, both granulocyte colony-stimulating factor (G-CSF) and interleukin-1ß(IL-1ß) in serum was found to show the same chronological change pattern between human and mice model. Next, we examined changes in cytokine expression in cirrhosis liver before and at 1, 2, 3, and 4 weeks after BMC infusion. Both G-CSF and IL-1ß were undetectable in the liver tissues before and at 1 week after BMC infusion but increased at 2 weeks and continued until 4 weeks after infusion. The infused BMCs induced an early decrease of both G-CSF and IL-1ß in serum and an increase in the model mice with LC. These dynamic cytokine changes might be important to repair liver cirrhosis after BMC infusion.

Fluorodeoxyglucose positron emission tomography response and normal tissue regeneration after stereotactic body radiotherapy to liver metastases.

PURPOSE: To characterize changes in standardized uptake value (SUV) in positron emission tomography (PET) scans and determine the pace of normal tissue regeneration after stereotactic body radiation therapy (SBRT) for solid tumor liver metastases. METHODS AND MATERIALS: We reviewed records of patients with liver metastases treated with SBRT to ≥40 Gy in 3-5 fractions. Evaluable patients had pretreatment PET and ≥1 post-treatment PET. Each PET/CT scan was fused to the planning computed tomography (CT) scan. The maximum SUV (SUV(max)) for each lesion and the total liver volume were measured on each PET/CT scan. Maximum SUV levels before and after SBRT were recorded. RESULTS: Twenty-seven patients with 35 treated liver lesions were studied. The median follow-up was 15.7 months (range, 1.5-38.4 mo), with 5 PET scans per patient (range, 2-14). Exponential decay curve fitting (r=0.97) showed that SUV(max) declined to a plateau of 3.1 for controlled lesions at 5 months after SBRT. The estimated SUV(max) decay half-time was 2.0 months. The SUV(max) in controlled lesions fluctuated up to 4.2 during follow-up and later declined; this level is close to 2 standard deviations above the mean normal liver SUV(max) (4.01). A failure cutoff of SUV(max) ≥6 is twice the calculated plateau SUV(max) of controlled lesions. Parenchymal liver volume decreased by 20% at 3-6 months and regenerated to a new baseline level approximately 10% below the pretreatment level at 12 months. CONCLUSIONS: Maximum SUV decreases over the first months after SBRT to plateau at 3.1, similar to the median SUV(max) of normal livers. Transient moderate increases in SUV(max) may be observed after SBRT. We propose a cutoff SUV(max) ≥6, twice the baseline normal liver SUV(max), to score local failure by PET criteria. Post-SBRT values between 4 and 6 would be suspicious for local tumor persistence or recurrence. The volume of normal liver reached nadir 3-6 months after SBRT and regenerated within the next 6 months.

Hepatic proliferation after partial liver irradiation in Sprague-Dawley rats.

The liver has powerful capability to proliferate in response to various injuries, but little is known as to liver proliferation after irradiation (IR) injury. This study investigated whether liver proliferation could be stimulated in low-dose irradiated liver by partial liver IR injury with high dose radiation. Sprague-Dawley rats were irradiated by 6-MV X-ray with single dose of 25 Gy to the right-half liver, while the left-half liver was shielded (0.7 Gy) or irradiated with single doses of 3.2, 5.6, and 8.0 Gy, respectively. Hepatic proliferation in the shielded and low-dose irradiated left-half liver was evaluated by serum hepatic growth factor (HGF), proliferating cell nuclei antigen (PCNA), liver proliferation index (PI), hepatocyte mitosis index (MI). The observation time was 0 day (before IR), 30, 60, 90, and 120 days after IR. Our results showed that serum HGF and hepatocyte HGF mRNA increased after IR with HGF mRNA peak on day 30 in the shielded and low-dose irradiated left-half livers, and their values increased as the dose increased to the left-half liver. Liver PI and PCNA mRNA peaked on day 60 with stronger expressions in higher doses-irradiated livers. MI increased after IR, with the peak noted on day 60 in the shielded and on day 90 in the low-dose irradiated left-half livers. There was a 30 day delay between MI peaks in the shielded and low-dose irradiated livers. In conclusion, 25 Gy partial liver IR injury could stimulate the shielded liver and low-dose irradiated liver to proliferate. In the livers receiving a dose range of 3.2-8.0 Gy, the proliferation was stronger in higher doses-irradiated liver than the low-dose irradiated. However, IR delayed hepatocyte mitosis.

Effect of laser on the remnant liver after the first 24 hours following 70 percent hepatectomy in rats / Efeito do laser no fígado remanescente nas primeiras 24 horas após hepatectomia a 70 por cento em ratos

PURPOSE: To evaluate the mitochondrial function of the remnant liver (RL) in the early phase of liver regeneration in rats after 70 percent partial hepatectomy (PH). METHODS: Sixty male Wistar rats (200-250g) submitted to 70 percent PH were divided into five groups according to the time of euthanasia and application or not of laser light: C = Control, time zero; 2 minutes, 4, 6 and 24 hours after PH. The dose of laser radiation was 22.5 J/cm², wavelength of 660 nm (visible/red), in the remnant liver. We studied the respiration activated by ADP (state 3), basal mitochondrial respiration (state 4), respiratory control ratio (RCR) and mitochondrial membrane potential (MMP). RESULTS: The mitochondrial function of RL changed at 4 and 6 hours after PH, with a significant increase in state 3 and a concomitant increase in state 4 and with maintenance of RCR. MMP differed significantly between the groups biostimulated with laser radiation and the control group 4 hours after HP, with a substantial reduction in the non-laser groups. CONCLUSION: The laser light at the dose used in this study did not induce additional damage to the RL and seems to have delayed the hepatocellular metabolic overload of the remnant liver.

OBJETIVO: Avaliar a função mitocondrial do fígado remanescente (FR) na fase precoce da regeneração hepática em ratos após hepatectomia parcial (HP) a 70 por cento. MÉTODOS: Sessenta ratos machos Wistar (200 - 250g) submetidos à HP a 70 por cento, foram distribuídos em cinco grupos de acordo com o tempo de eutanásia e com aplicação ou não de luz laser: C= Controle,tempo zero; 2 minutos, 4, 6 e 24 horas após HP. O laser foi utilizado na dose 22.5 J/cm², 660 nm, no FR.Estudou-se o estado 3 (respiração ativada por ADP), estado 4 (respiração mitocondrial basal), razão de controle respiratório,estado 3/estado 4 (RCR) e o potencial de membrana mitocondrial(PMM). RESULTADOS: A função mitocondrial do FR alterou-se no período de 4 e 6 horas após a HP com aumento significativo do estado 3 e aumento concomitante do estado 4, com manutenção da RCR. O PMM apresentou diferença significativa entre os grupos bioestimulados com laser e o controle a partir de 4 horas pós HP, com queda importante do grupo sem laser e tendência a equiparação dos valores após 24 horas. CONCLUSÃO: A luz laser, na dose utilizada no presente estudo, não induziu lesão adicional ao FR e parece ter retardado a sobrecarga hepatocelular do fígado remanescente.

Effect of laser on the remnant liver after the first 24 hours following 70% hepatectomy in rats.

PURPOSE: To evaluate the mitochondrial function of the remnant liver (RL) in the early phase of liver regeneration in rats after 70% partial hepatectomy (PH). METHODS: Sixty male Wistar rats (200-250g) submitted to 70% PH were divided into five groups according to the time of euthanasia and application or not of laser light: C = Control, time zero; 2 minutes, 4, 6 and 24 hours after PH. The dose of laser radiation was 22.5 J/cm(2), wavelength of 660 nm (visible/red), in the remnant liver. We studied the respiration activated by ADP (state 3), basal mitochondrial respiration (state 4), respiratory control ratio (RCR) and mitochondrial membrane potential (MMP). RESULTS: The mitochondrial function of RL changed at 4 and 6 hours after PH, with a significant increase in state 3 and a concomitant increase in state 4 and with maintenance of RCR. MMP differed significantly between the groups biostimulated with laser radiation and the control group 4 hours after HP, with a substantial reduction in the non-laser groups. CONCLUSION: The laser light at the dose used in this study did not induce additional damage to the RL and seems to have delayed the hepatocellular metabolic overload of the remnant liver.

Hepatic regeneration after sublethal partial liver irradiation in cirrhotic rats.

Our previous animal study had demonstrated that partial liver irradiation (IR) could stimulate regeneration in the protected liver, which supported the measurements adopted in radiotherapy planning for hepatocellular carcinoma. The purpose of this present study is to investigate whether cirrhotic liver repopulation could be triggered by partial liver IR. The cirrhosis was induced by thioacetamide (TAA) in rats. After cirrhosis establishment, TAA was withdrawn. In Experiment 1, only right-half liver was irradiated with single doses of 5 Gy, 10 Gy and 15 Gy, respectively. In Experiment 2, right-half liver was irradiated to 15 Gy, and the left-half to 2.5 Gy, 5 Gy and 7.5 Gy, respectively. The regeneration endpoints, including liver index (LI); mitotic index (MI); liver proliferation index (LPI); PCNA-labeling index (PCNA-LI); serum HGF, VEGF, TGF-α and IL-6, were evaluated on 0 day, 30-day, 60-day, 90-day, 120-day and 150-day after IR. Serum and in situ TGF-ß1 were also measured. In both experimental groups, the IR injuries were sublethal, inducing no more than 9% animal deaths. Upon TAA withdrawal, hepatic regeneration decelerated in the controls. In Experiment 1 except for LI, all other regeneration parameters were significantly higher than those in controls for both right-half and left-half livers. In Experiment 2 all regeneration parameters were also higher compared with those in controls for both half livers. Serum HGF and VEGF were increased compared with that of controls. Both unirradiated and low dose-irradiated cirrhotic liver were able to regenerate triggered by sublethal partial liver IR and higher doses and IR to both halves liver triggered a more enhanced regeneration.

Enhanced liver regeneration following acute hepatectomy by low-level laser therapy.

OBJECTIVE: The aim of the present study was to investigate the effect of low-level laser therapy (LLLT) on liver regeneration following hepatectomy. BACKGROUND: LLLT has been found to modulate various biological processes. MATERIALS AND METHODS: Twelve mature male rats were used. The liver was exposed, and 70% of it was excised. The rats were assigned randomly to two groups: control, non-laser treated, and experimental, laser-treated (diode [Ga-Al-As] laser 804 nm) group. For determination of newly formed blood vessels and proliferating cells, 5-Bromo-2'deoxyuridine (BrdU) was injected intraperitoneally. The rats were sacrificed 2 d post hepatectomy, and histological sections from each liver were processed for analysis of new blood-vessel formation using BrdU immunostaining kit. Mesenchymal stem cells (MSCs) were assessed using c-kit immunostaining. BrdU-labeled cells were counted as for estimation of newly formed hepatic cells. RESULTS: It was found that the number of proliferating cells (BrdU positive cells) per area in the regenerating regions of the livers were significantly (p < 0.01) 2.6-fold higher in the laser-treated rats than in the control non-laser-treated rats. The density of the newly formed blood vessels and c-kit immunopositive cells in the regenerating area of the laser-treated livers was significantly (p < 0.01) 3.3- and 2.3-fold respectively higher than the control non-laser treated livers. CONCLUSION: It is concluded that LLLT following acute hepatectomy most probably stimulates a significant enhancement of liver regeneration conducive to both the formation of new hepatocytes and MSCs and angiogenesis in the regenerating liver.

Liver sinusoidal endothelial and biliary cell repopulation following irradiation and partial hepatectomy.

AIM: To investigate whether irradiation (IR) and partial hepatectomy (PH) may prepare the host liver for non-parenchymal cell (NPC) transplantation. METHODS: Livers of dipeptidyl peptidase IV (DPPIV)-deficient rats were pre-conditioned with external beam IR (25 Gy) delivered to two-thirds of the right liver lobules followed by a one-third PH of the untreated lobule. DPPIV-positive liver cells (NPC preparations enriched for liver sinusoidal endothelial cells (LSECs) and hepatocytes) were transplanted via the spleen into the recipient livers. The extent and quality of donor cell engraftment and growth was studied over a long-term interval of 16 wk after transplantation. RESULTS: Host liver staining demonstrated 3 different repopulation types. Well defined clusters of donor-derived hepatocytes with canalicular expression of DPPIV were detectable either adjacent to or in between large areas of donor cells (covering up to 90% of the section plane) co-expressing the endothelial marker platelet endothelial cell adhesion molecule. The third type consisted of formations of DPPIV-positive duct-like structures which co-localized with biliary epithelial CD49f. CONCLUSION: Liver IR and PH as a preconditioning stimulus enables multiple cell liver repopulation by donor hepatocytes, LSECs, and bile duct cells.

Regenerative capacity of normal and irradiated liver following partial hepatectomy in rats.

PURPOSE: To investigate the regenerative capacity and proliferation related to cell cycle modulators in irradiated livers after partial hepatectomy (PH) in rats. METHODS AND MATERIALS: Two experimental groups were given a single dose of either 4-Gy or 8-Gy photon radiation to the whole liver following PH. The control group underwent only PH, without irradiation. The liver specimens were analysed for apoptosis, proliferation and cell cycle related genes between 0.5 and 12 days. RESULTS: Mean change in weight of the remnant liver in the 8-Gy group was significantly lower than in the control and 4-Gy groups. The apex of proliferating cell nuclear antigen labelling and bromodeoxyuridine incorporation index in two irradiated groups were also apparently lower than that in control group. After PH, transforming growth factor beta-1 (TGFbeta1), and the type II receptor of TGFbeta (TGFbetaR-II), anti-tumour protein 53(p53) and anti-tumour protein21(p21) protein expression in the irradiated livers was higher than in unirradiated ones. Significant apoptosis was noted in 8-Gy group. However, the maximal value of hepatocyte growth factor (HGF) mRNA and protein expression in the irradiated group was suppressed and restoration of liver function was delayed. CONCLUSION: Whole liver lower dose irradiation can attenuate regenerative capacity following partial hepatectomy in rats.

Does adjuvant radiotherapy suppress liver regeneration after partial hepatectomy?

PURPOSE: To analyze the influence of the adjuvant radiotherapy (RT) on the liver regeneration and liver function after partial hepatectomy (PH). METHODS AND MATERIALS: Thirty-four patients who underwent PH for biliary tract cancer between October 2003 and July 2005 were reviewed. Hemihepatectomy was performed in 14 patients and less extensive surgery in 20. Of the patients, 19 patients had no adjuvant therapy (non-RT group) and 15 underwent adjuvant RT by a three-dimensional conformal technique (RT group). Radiation dose range was 40 to 50 Gy (median, 40 Gy). Liver volume on computed tomography and the results of liver function tests at 1, 4, 12, 24, and 52 weeks after PH were compared between the RT and non-RT groups. RESULTS: The preoperative characteristics were identical for both groups. During the interval between Weeks 4 and 12 when adjuvant RT was delivered in the RT group, the increase in liver volume was significantly smaller in the RT group than non-RT group (22.9 +/- 38.3cm(3) and 81.5 +/- 75.6cm(3), respectively, p = 0.007). However, the final liver volume measured at 1 year after PH did not differ between the two groups (p = 0.878). Liver function tests were comparable for both groups. The resection extent and original liver volume was independent factors for final liver volume measured at 1 year after PH. CONCLUSIONS: In this study, adjuvant RT delayed the liver regeneration process after PH, but the volume difference between the two study groups became nonsignificant after 1 year. Adjuvant RT had no additional adverse effect on liver function after PH.

The use of light-emitting diodes to stimulate mitochondrial function and liver regeneration of partially hepatectomized rats.

The biostimulating effect of laser radiation has been observed in many areas of Medicine. However, there are still several questions to be answered, among them the importance of light coherence in the stimulatory process. In the present study, we used light-emitting diodes (LED) to promote the stimulation of liver regeneration after partial hepatectomy in rats. Fourteen male Wistar rats weighing 200-250 g were submitted to partial hepatectomy (70%) followed by LED light irradiation (630 nm) of the remaining part of the liver at two doses, i.e., 10 (N = 7) and 140 (N = 7) J/cm(2). A group irradiated with laser, 590 nm (N = 7, 15 J/cm(2)) was performed for the study of proliferating cell nuclear antigen-labeling index. Data are reported as mean +/- SEM. Statistical comparisons of the groups were performed by analysis of variance for parametric measurements followed by the Bonferroni post-test, with the level of significance set at P < 0.05. Respiratory mitochondrial activity was increased in the irradiated groups (states 3 and 4; P < 0.05), with better results for the group exposed to the lower LED dose (10 J/cm(2)). The proliferating cell nuclear antigen-labeling index, by immunohistochemical staining, was similar for both LED-exposed groups (P > 0.05) and higher than for the control group (P < 0.05). The cell proliferation index obtained with LED and laser were similar (P > 0.05). In conclusion, the present results suggest that LED irradiation promotes biological stimulatory effects during the early stage of liver regeneration and that LED is as effective as laser light, independent of the coherence, divergence and cromaticity.