The influence of nuclear radiation in expression of CD4 using immunohistochemical features in some organs of rats infested with Echinococcus granulosus and assessment of liver enzymes

1. Low energy of nuclear particles with increasing duration time reduces the viability of protoscoleces.
2. Nuclear particles showed significantly reduced enzyme levels (GOT, GPT and ALP) correlating with decreased parasite burden and improved liver function.
3. Nuclear particles induced immune response and enhanced CD4 expression which is varied in the liver, spleen and intestine according to infection duration and radiation exposure.

Introduction

Hydatid disease or echinococcosis is an illness transmitted by Echinococcus spp. that brings about health and financial challenges worldwide especially in areas relying heavily on animal husbandry for their livelihoods (1). Canids are the definitive host of the adult stage of Echinococcus spp. The infection occurs when hosts ingest eggs of the parasite and develop into the larval stage, hydatid cyst,which found in the most organs of domestic animals such as cattle, sheep and camels this cyst grows slowly from inside to outside, it consists germinal layer and laminated layer and filled with hydatid cyst fluid which results from the secretion of soluble components by the germinal layer and may also provide nutrients necessary for the growth and development of hydatid cysts and of the protoscoleces that are collected within the hydatid cyst fluid, these protoscoleces will generate the adult worm in definitive hosts. The infection mainly targets the liver and lungs by forming hydatid cysts that can disrupt organ function and result in complications (2). Zai et al. (3) demonstrated that liver is the main target organ for hydatidosis. The larval stage of Echinococcosis lead to formation of fibrotic cysts which lead to develop a histological barrier between healthy liver tissues and damaged that decrease host’s inflammatory defenses against protoscoleces and support the protoscoleces to invasion in the liver (4-7). Hepatic damage is accompanied with changes in the level liver function enzymes like majority of especially GPT, GOT and ALP. The level of liver enzyme are depended on the value of harm or necrosis in hepatic tissue. A raise in liver enzymes are showing various kinds of liver damage (8). The body's defense against Echinococcus infection includes pathways that activate and regulate T cell subsets like CD4+ for managing the immune response against the parasite (9). There has been a rising interest in the potential uses of nuclear particle in parasitology especially alpha particles due to its ability to deactivate harmful agents (10). Alpha particles possess an energy transfer capability that allows them to inflict significant harm on biological tissues and pathogens over short distances (11-13). Nevertheless, the effects of this type of radiation on host immune reactions such as CD4 protein levels and the specific impacts on different organs have not been thoroughly investigated yet (14).

Moreover, the impact of radiation on liver function affected by echinococcosis requires exploration to evaluate possible treatment benefits or side effects. The current study focused on examining how nuclear particle radiation impacts the survival of Echinococcus protoscoleces and the related physiological and immune reactions in rats as a model organism. This study aims to understand the relationship between radiation exposure and its effects on liver function enzymes and CD4 expression in organs in the context of hydatid disease. The results could enhance our knowledge of using radiation-based treatments for infections and their implications for overall host well-being.

Materials and methods

Ethical approval

Ethical approvals references number UM.VET.2024.126, dated January 2, 2024 for handling animals was awarded by institutional Animal Care and Use Committee in College of Veterinary Medicine, University of Mosul, Mosul, Iraq.

Sample collection and viability assessment

Hydatid cysts were obtained from the sheep livers at the slaughterhouse in Mosul City. Then checked promptly to see if they contained protoscoleces by using an aqueous eosin dye (0.1%) to distinguish between live (green colored ones area) and dead (red colored ones) under a microscope. The movement of the protoscoleces was regarded as an indicator of viability with samples showing 98%- 99% viability being selected for analysis according to Smith and Barret method (15).

Isolation of protoscoleces

According to Smyth (16) cysts were sterilized externally using 1% alcoholic iodine solution and punctured using a sterile 21G needle to aspirate cyst fluid into a 20 mL syringe. The fluid was centrifuged at 3000 rpm for three cycles each time adding penicillin (2000 IU) and streptomycin (1 g/L) to the wash solution (PBS, pH 7.2). Viability of protoscoleces were suspended in sterile PBS and stored for further experiments.

Viability calculation

A 20 µL protoscolex suspension was mixed with an equal volume of 0.1% eosin dye and examined microscopically. The viability percentage was calculated as follows viability (%)= (number of liver protoscoleces/total protoscoleces)*100. The assessment was performed three times and the mean viability was recorded (16).

Radiation exposure of protoscoleces

Protoscoleces were exhibited to nuclear particles Americium-241 (²⁴¹Am) at two energy (2.3 and 4.3MeV) for varying durations (17). For each duration 100 µL of protoscoleces were irradiated. At 4.3MeV: Exposure durations were 5, 20, 45, 60, and 90 minutes. At 2.3MeV: Exposure durations were 10, 30, 50, 60, and 90 minutes. Post-exposure, the percentage of viable and dead protoscoleces was calculated microscopically in the nuclear laboratory in the college of Education for Pure Science by using Americium-241 (²⁴¹Am) source in the activity (1µCi).

Experimental design

Thirty-five adult rats were divided into seven groups (n=5) per group. Group 1 rats were used as negative control. Group 2 rats injected, intraperitoneally, with protoscoleces irradiated for 60 minutes then sacrificed after 4 months. Group 3 rats injected, intraperitoneally, with protoscoleces irradiated for 60 minutes then they sacrificed after 5 months. Group 4 rats injected, intraperitoneally, with protoscoleces irradiated for 90 minutes then sacrificed after 4 months. Group 5 rats injected, intraperitoneally, with protoscoleces irradiated for 90 minutes then sacrificed after 5 months. Group 6 (Positive control) rats injected, intraperitoneally, with non-irradiated protoscoleces and they sacrificed after 4 months. Group 7 (Positive control) rats injected, intraperitoneally, with non-irradiated protoscoleces then sacrificed after 5 months.

Immunohistochemical

Immunohistochemical analysis of CD4 expression were used according to Alturkistani et al. (18). Preparation of tissues, liver and spleen samples were collected after euthanizing rats then fixed in 10% neutral buffered formalin and embedded in paraffin. 5µm for both samples were prepared utilizing a microtome.

Immunohistochemical staining

Slides were removed in xylene and rehydrated estimated using different levels of alcohols. Antigen recovery was completed by a citrate buffer (pH 6.0) at 95°C for twenty minutes. The activity of endogenous peroxidase was plugged via incubating divisions for 3% H2O2. CD4 expression was appreciated semi-quantitatively established upon staining density and the proportion of positively stained cells. compared Results with experimental sets. Slides had been incubated overnight at 4°C with a monoclonal anti-CD4 antibody. After washing, sections had been incubated with a biotinylated antibody, streptavidin-HRP. The chromogenic material application di-aminobenzidine (DAB). Slides were dyed by hematoxylin, desiccated, and fixed.

Weight and hypertrophy index of liver and spleen

Liver and spleen hypertrophy index were estimated according to Kroeze and Tanner (19) as follows: organ index = (organ weight/(body weight-organ weight)*100.

Liver function tests

Serum was analyzed for liver enzymes (GOT, GPT, and ALK) depending on El-Lehieh et al. (20).

Statistical analysis

One-way Analysis of Variance (ANOVA) was used to test the differences between groups followed by Tukey's post-hoc test for multiple comparisons to determine statistically significant differences among the groups. The level of statistical significance was set at (p < 0.05) with values below this threshold considered indicative of significant differences between the groups. The results were presented as mean±standard deviation to illustrate the variation within each group (21).

Results

The data in Table 1 illustrate the effects of nuclear particles irradiation on living protoscoleces at energy 4.3MeV with varying exposure durations (Table 1). The data presented in Table 2 illustrates the killing rates of living protoscoleces exposed to nuclear particles at 2.3MeV energy over varying durations. The results indicate a significant relationship between exposure time and killing efficiency.

Table 1: Killing rate of living protoscoleces by nuclear particles at energy 4.3MeV at different exposure durations

Table 2: Killing rate of living protocoleces by nuclear particles at 2.3MeV energy at different exposure durations

The table 3 shows liver and spleen weights and hypertrophy index after five months of exposure to different conditions. Liver weight, the positive control group showed a higher liver weight (3.78±0.1) compared to the 60minutes radiation group (2.86±0.06) and the 90minutes radiation group (2.92±0.07). The p-value is < 0.001 indicating a highly significant difference between the positive control group and the other groups. Hypertrophy Index (Liver), the positive control group had the highest liver hypertrophy index (68.4±1.03) compared to the other groups (47±1.51 and 49.8±1.06). The p-value is < 0.001 suggesting a highly significant difference. Spleen weight, the positive control group showed a higher spleen weight (2.44±0.13) compared to the other groups (1.84±0.08 and 1.94±0.11). The p-value is 0.002 indicating a significant difference between the groups. Hypertrophy Index (Spleen), the positive control group had the highest spleen hypertrophy index (39±4.4) compared to the other groups (27.8±1.88 and 29.8±1.24). The p-value is 0.037 showing a significant difference between the groups.

Table 3: Liver and spleen weights and their hypertrophy index after five months of exposure

Data expressed as Mean±standard error, (no. rats = 5). *There is a significant difference from other groups at p≤0.05.

The table 4 shows the effects of nuclear particle exposure on liver enzymes (GPT, GOT, and ALP) after four months of exposure. The data is presented as Mean±Standard Error and the reference ranges for the enzymes are provided. Glutamate Pyruvate Transaminase (GPT), the positive control group showed significantly higher GPT levels (56.2±13.3U/L) compared to the radiation exposure groups (23.2±2.59U/L for 60 minutes and 20.2±5.93U/L for 90 minutes). The p-value is 0.031 indicating a statistically significant difference between the positive control group and the radiation exposure groups. The positive control group's GPT range (4-44) indicating elevated enzyme levels. Glutamate Oxaloacetate Transaminase (GOT), the positive control group showed higher GOT levels (156.8±37.55U/L) compared to both radiation exposure groups (70.4±5.95U/L for 60 minutes and 62.21±5.46U/L for 90 minutes). The p-value is 0.034 indicating a significant difference. The positive control group's GOT levels are elevated compared to the GOT range (8-38) indicating liver stress. Alkaline Phosphatase (ALP), the positive control group showed significantly higher ALP levels (381.8±18.16U/L) compared to both radiation exposure groups (126±11.33U/L for 60 minutes and 23.4±18.82U/L for 90 minutes). The p-value is <0.001 indicating a highly significant difference between the groups. The positive control group’s ALP levels are well above the ALP range (114-338) suggesting liver dysfunction.

Table 4: Effect of nuclear particles on liver enzymes after four months of exposure

Histopathological examination

Figure 1shows rat's liver of hydatid cyst infected (positive control) group without radiation, [A]: after 4 months and [B]: after 5 months shows presents of laminated membrane of protoscoleces (double head arrow), surrounding by increased fibrous tissue (black arrow), infiltration of inflammatory cells in portal area (green arrow) and necrosis of hepatocytes (blue arrow) with increased fibrous tissue between it (yellow arrow) [A and B]. After 4 months of infected rats with protoscoleces exposure to nuclear particles for 60minutes mild necrosis and vacuolar degeneration of the hepatocytes, stenosis of sinusoids and mild infiltration of inflammatory cells were recorded [A]. On the other hand, mild necrosis of the hepatocytes, dilatation of sinusoids were observed after 4 months of infected rats with protoscoleces exposure to nuclear particles for 90minutes [B] (Figure 2). Figure 3 shows very mild vacuolar degeneration of hepatocytes and congestion of central vein [A] after 5 months of infected rats with protoscoleces exposure to nuclear particles for 60minute. On the other hand, normal architecture of hepatic tissue, central vein and portal area were seen after 5 months of infected rats with protoscoleces exposure to nuclear particles for 90 minutes [B].

Figure 1: photomicrograph of rat's liver of hydatid cyst infected (positive control) group, [A]: after 4 months and [B]: after 5 months shows presents of laminated membrane of hydatid cyst (double head arrow), surrounding by increased fibrous tissue (black arrow), infiltration of inflammatory cells in portal area (green arrow) and necrosis of hepatocytes (blue arrow) with increased fibrous tissue between it (yellow arrow). 100X.

Figure 2: photomicrograph of rat's liver. [A]: After 4 months of infected rats with protoscoleces exposure to nuclear particles for 60minutes: mild necrosis (black arrow) and vacuolar degeneration of the hepatocytes (green arrow), stenosis of sinusoids (blue arrow) and mild infiltration of inflammatory cells (yellow arrow). [B]: after 4 months of infected rats with protoscoleces exposure to nuclear particles for 90minutes: mild necrosis of the hepatocytes (black arrow), dilatation of sinusoids (green arrow).

Figure 3: photomicrograph of rat's liver. [A]: after 5 months of infected rats with protoscoleces exposure to nuclear particles for 60minute: very mild vacuolar degeneration of hepatocytes (black arrow), and congestion of central vein (green arrow). [B]: after 5 months of infected rats with protoscoleces exposure to nuclear particles for 90minutes: intact architecture of hepatocytes (black arrow), central vein (green arrow) and portal area (blue arrow). 100X.

Immunohistochemical examination

Immunohistochemical expression of CD4 of the negative control groups were shown in figure 4. [A]: Liver; [B]: spleen, [C]: intestine. Immunohistochemical analysis of CD4 expression in the liver, spleen, and intestine of rats infected with hydatid cysts showed varying intensities across different treatment durations and tissues. In the liver (Figure 5), CD4 expression in the 4-month infected group demonstrated very intense expression in the 60minutes radiation treatment group [A] and intense expression in the 90minutes radiation treatment group [B]. In the 5-month infected group, moderate expression was observed in the 60minutes treatment group [C], while weak expression was detected in the 90minutes treatment group [D]. Similarly, in the spleen (Figure 6), the 4-month infected group displayed very intense expression of CD4 in the 60minutes radiation treatment group [A] and intense expression in the 90minutes group [B]. In the 5-month infected group, CD4 expression was moderate in the 60minutes treatment group [C] and weak in the 90minutes group [D]. In the intestine (Figure 7) the 4-month infected group exhibited very intense expression of CD4 in the 60minutes radiation treatment group [A] and intense expression in the 90minutes group [B]. In the 5-month infected group, moderate expression was observed in the 60minutes treatment group [C] and weak expression was noted in the 90minutes group [D]. All samples were examined at 100X magnification, and had a scale bar of 100 µm.

Figure 4: Immunohistochemical expression of CD4 of the negative control. [A]: Liver; [B]: spleen, [C]: intestine. They reveal weak expression. 100X. scale-bar=100µm.

Figure 5: Immunohistochemical expression of CD4 in the Rat's liver. [A& B]: 4 months and [C& D]: 5 months hydatid cyst infected. [A]: 60minutes radiation treatment group: very intense expression (arrows). [B]: 90minutes radiation treatment group: intense expression (arrows). [C]: 60minutes radiation treatment group: moderate expression (arrows). [D]: 90minutes radiation treatment group: weak expression (arrows); 100X. scale-bar=100µm.

Figure 6: Immunohistochemical expression of CD4 in the Rat's spleen. [A& B]: 4 months and [C& D]: 5 months hydatid cyst infected. [A]: 60minutes radiation treatment group: very intense expression (arrows). [B]: 90minutes radiation treatment group: intense expression (arrows). [C]: 60minutes radiation treatment group: moderate expression (arrows). [D]: 90minutes radiation treatment group: weak expression (arrows) 100X. scale-bar=100µm.

Figure 7: Immunohistochemical expression of CD4 in the Rat's intestine. [A& B]: 4 months and [C& D]: 5 months hydatid cyst infected. [A]: 60minutes radiation treatment group: very intense expression (arrows). [B]: 90minutes radiation treatment group: intense expression (arrows). [C]: 60minutes radiation treatment group: moderate expression (arrows). [D]: 90minutes radiation treatment group: weak expression (arrows) 100X. scale-bar=100µm.

Discussion

Hydatid disease known as echinococcosis also known as cystic echinococcosis is a neglected zoonotic illness brought on by Echinicoccus spp. (21,22). The present study investigated the therapeutic effects of nuclear particle on hydatid cyst and its impact on immune responses, organ hypertrophy index, liver enzyme levels, and histological changes of tissues in infected rats. The findings emphasize the dual role of radiation in reducing parasite viability and modulating host immune and physiological responses, these results agree with the findings of other studies (23,24). Radiation effectively reduced protoscolex viability in an energy and duration-dependent manner. A 90-minute exposure at 4.3 MeV achieved an 87% killing rate, while at 2.3 MeV the rate increased to 95%. These results consistent with that of Raziani et al. (25) who highlight the efficacy of alpha particles in damaging parasite DNA and cellular structures. Other researchers demonstrated that molecular level and alpha particles directly damage the DNA of parasitic cells by inducing double-strand breaks in the DNA, these breaks are highly detrimental to the cells especially if not properly repaired. Additionally, alpha particles generate reactive molecules such as reactive oxygen species (ROS) which cause further damage to cellular membranes, proteins, and DNA (26-28). The increased effect with reduced distance is due to the higher linear energy transfer (LET) to the cells which raises the likelihood of causing lethal damage (29). Moreover, increased exposure duration results in cells receiving higher radiation doses enhancing cumulative damage and reducing survival chances (30). The observed efficiency at 2.3 MeV suggests prolonged exposure compensates for reduced radiation intensity at greater distances, aligning with previous findings on radiation’s cytotoxicity in parasitic models (31). In the current study rats in the positive control group exhibited significant liver and spleen hypertrophy index, indicative of the metabolic and immune burden imposed by hydatid cyst infection. Radiation treatment reduced organ weights and hypertrophy index with significant differences noted (p < 0.001 for liver; p = 0.037 for spleen). These results are in agreement with Szewczyk-Golec's et al. (32) and Eissa et al. (33) findings which suggested that the reduction in organ weight and hypertrophy index decreased parasite viability and associated pathological effects reflecting the therapeutic potential of radiation in alleviating organ stress.

In the present study, infected rats with protoscoleces without radiation treatment displayed elevated GPT, GOT, and ALP levels indicating liver damage and biliary dysfunction. Radiation-treated groups showed significantly reduced enzyme levels (p = 0.031 for GPT, p = 0.034 for GOT, p < 0.001 for ALP), correlating with decreased parasite burden and improved liver function. The group exposed to radiation for 60 minutes showed decreased enzyme levels in comparison to the group exposed for 90 minutes indicating that extended exposure could worsen tissue stress even as it decreases infection levels. Ilderbayev et al. (34); Benchabane et al. (35) suggest that variation in enzyme levels may be clarified by the rise in radicals and oxidative stress due to prolonged exposure to radiation leading to tissue damage Heightened local inflammation. Conversely the decrease in infections could be attributed to the improved efficiency of radiation in eliminating parasite with exposure periods. However, this effectiveness may compromise the integrity of tissues (36). The results underscore the significance of striking an equilibrium between the length of radiation exposure and its healing advantages versus its effects since extended exposure could lead to negative tissue outcomes that overshadow the gains in infection reduction.

The examination of tissue samples showed liver damage in the test group that received treatment, then radiation therapy due to conditions like cell death in the hepatocytes and abnormal changes in cell structure and function along with decreased blood flow and blockage, in central veins caused by inflammation. In the group treated for 60 minutes on infected subjects after 120 days of infection observations indicated some tissue damage and narrowing of blood vessels; however, in the group treated for 90 minutes there was an increase, in the activity of Kupffer cells suggesting a response leading to clearance of parasites. Groups infected for 60 minutes displayed degeneration and congestion, in the central vein after 6 months of infection. Notably, the 90 minutes group exhibited intact liver architecture, reflecting effective parasite control and tissue recovery. These improvements highlight radiation’s ability to restore liver integrity over time, with longer exposure durations demonstrating greater efficacy in mitigating histopathological damage (37,38).

In the present study, CD4 expression in the liver, spleen, and intestine varied with infection duration and radiation exposure. After 4 months of injected rats with protoscoleces exposed to nuclear particles for 60 minutes, exhibited very intense CD4 expression across tissues, while those treated for 90 minutes showed intense expression. At 5 months, CD4 expression decreased to moderate (60 minutes treatment) and weak (90 minutes treatment) levels. In Liver, Intense CD4 expression at 4 months reflects an active immune response to infection. The decline at 5 months correlates with reduced parasite burden and diminished immune stimulation suggesting tissue recovery and immune regulation (39). In Spleen, Similar patterns in CD4 expression indicate systemic immune modulation. The reduced expression at 5 months highlights the therapeutic efficacy of prolonged radiation in suppressing parasitic antigens (40,41). In Intestine, CD4 trends mirrored other tissues, with reduced expression at 5 months indicating localized immune resolution and potential tissue recovery. These results show an interplay, between boosting the response and controlling it to prevent infection and reduce long term inflammation.

Conclusion

The present study investigated the therapeutic effects of nuclear particle on hydatid cyst and its impact on immune responses, organ hypertrophy index, liver enzyme levels, and histological changes of tissues in infected rats. Low energy of nuclear particles with increasing duration time reduces the viability of protoscoleces. Nuclear particles showed significantly reduced enzyme levels (GPT, GOT and ALP) correlating with decreased parasite burden and improved liver function. Nuclear particles induced immune response and enhanced CD4 expression which is varied in the liver, spleen and intestine according to infection duration and radiation exposure. These findings emphasize the dual role of radiation in reducing protoscoleces viability and modulating host immune and physiological responses, these discoveries lay the groundwork, for exploring how radiation therapy can be applied in treating illnesses.

Acknowledgment

The researchers extend their gratitude to the University of Mosul, College of Education for Pure Science for supporting the present study.

Conflicted interest

None

Editorial board note

Muneer S. Al-Badrany, Dhafer M. Aziz, and Raad A. Al-Sanjary, the editors of the Iraqi Journal of Veterinary Sciences, did not participate in any stage of the decision-making process for this article.