Age-associated changes in the lymphoid tissues of Boa constrictor

Abstract

Aging is a complex and multifaceted biological process that results in the gradual decline of physiological functions over time. It is associated with reduced performance across multiple systems, affecting metabolic, reproductive, musculoskeletal, and immune functions. While immune aging has been extensively studied in endothermic animals, and in particular mammals such as laboratory rodents, comparatively little is known about how aging manifests in ectothermic vertebrates like reptiles. This study explored the lymphoid tissue (spleen and thymus) of Boa constrictor, a boid snake indigenous to South and Central America and Mexico, but widely kept in captivity all over the world, for potential age-related changes. We observed a significant decrease in cellularity in the spleen, coupled with an increase in organ size correlated with age. In both spleen and thymus the connective tissue of capsule and trabeculae increased significantly with age, indicative of progressive fibrosis. In addition, several changes were observed with increasing frequency in older animals, epithelial hyperplasia in the thymic medulla as well stromal fibrosis and an increasing infiltration by so-called granular cells in both organs. Granular cells likely represent a leukocyte subtype; their presence indicates a progressive chronic low-grade inflammatory state in the lymphoid organs, a feature known as inflammaging in other animal classes. They may also play a role in the progressive fibrosis of the connective tissue. The results firstly describe morphological evidence of aging in B. constrictor and indicate similarities in the aging across animal classes.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12979-025-00519-7.

Background

Aging is a complex biological process characterized by the progressive decline of physiological functions, dysregulation of immune function and increasing cellular damage [1, 2]. While the relationship between aging and immune function is well established in mammals (e.g., humans, dogs, mice), it remains poorly understood in other vertebrates, where age-associated declines in immune function, also referred to as immunosenescence, have received little attention [2, 3]. Aging of reptiles has generally not been a focus of research, however, recently, the interest in comparing the longevity of endotherms (e.g., mammals) and ectotherms (e.g. reptiles) has increased [4–6]. This broadening of interest is driven by the fact that ectotherms hold many animal longevity and reproductive activity records, which highlights their value to improve the understanding of the mechanisms underlying lifespan extension and age-related physiological processes [4, 7]. It is important to note that, from an evolutionary perspective, reptiles are the sole surviving ectothermic amniotes and represent a link between ectothermic anamniotic fish and amphibians, and endothermic amniotic birds and mammals [8].

The reptile immune system has several features in common with its mammalian counterpart; this refers to certain components of both the innate (e.g., complement system, lysozymes, antimicrobial peptides) and adaptive immune response (e.g. T cell receptors, antibody production) that have been described at molecular and/or functional level in various reptile species [9]. However, research has also highlighted fundamental differences between the immune responses of mammals and reptiles. A notable example is the adaptive immune response in reptiles, which appears to be less specific than that of mammals [9]. Indeed, reptiles require substantially more time for antibody release and appear to generate very limited immunological memory [9, 10]. Moreover, reptiles have neither lymph nodes nor a Bursa Fabricii, although in some species specialized lymphoid structures, such as oesophageal tonsils in boid snakes [11]and lymphoid axillar structures in geckos [12], have been described. The reptilian spleen, which would hence be the expected site of B cell proliferation, activation and differentiation, lacks germinal centers; indeed, the most relevant parts of the B cell driven component of the specific immune response are still obscure in reptiles [9, 13, 14].

The boa constrictor (B. constrictor), a non-venomous boid snake native to Central and South America, is one of the most popular reptile species in the pet trade, primarily due to its adaptability to environmental conditions and relatively low-maintenance husbandry requirements [15]. It is also a snake species known for its rather long lifespan, averaging approximately 20 years in the wild and reaching 25 to 35 years in captivity [16, 17].

In a recent in depth study, we described the morphology and composition of the haemolymphatic tissues in B. constrictor [14]. To characterize the cellular components in situ, we applied immunohistochemistry to detect T cells and macrophages, RNA-in situ hybridization to highlight B cells, and special stains for connective tissue components such as collagen and reticulin. Our findings indicated that the spleen of B. constrictor is predominantly composed of T cells and is devoid of a red pulp [14]. The thymus was found to persist well beyond the age of sexual maturity; however, it exhibited an age-related decline in total cellularity, resembling the thymic involution observed in many mammals [14]. Since we observed anecdotal, potential age-related changes in the studied cohort but could not find much information on ageing and age-related features of the immune system of reptiles, we decided to undertake a more in-depth morphological study on thymus and spleen over the lifespan of boa constrictors.

Materials and methods

Study animals

The study was retrospectively undertaken on 48 B. constrictor from private owners, breeding collections and a reptile shelter. All animals had been submitted for a diagnostic postmortem examination by the owner; the latter was performed upon the owners’ request. Most boas (n = 44) had been submitted for euthanasia, the remaining two (Nos 14, 15) had died naturally and were examined to determine the cause of death.

The majority of the snakes originated from collections in which Boid Inclusion Body Disease (BIBD) had previously been diagnosed in one or several animals. The owners submitted the snakes to gain information on the general health status of the collection across age and sex groups (presence of BIBD/reptarenavirus infection or other viral/bacterial diseases of which the owners might not have been aware). Twenty animals (10.1 to 10.20) originated from a reptile shelter, a Switzerland-based volunteer organization that accepts reptiles from private owners or breeders who are unable to maintain their collection. These snakes were euthanized as the shelter had not succeeded in rehoming the animals within a prolonged period (years). For these individuals, information on their exact origin was not available.

For the present study only animals without significant pathological changes in the internal organs were chosen, to exclude any underlying diseases that could have affected the composition of the lymphatic tissue. Information on individual animals is provided in Table S1.

Euthanasia, gross and histological examination

All snakes submitted alive (n = 46) were euthanized by veterinarians according to the ASPA, (Animals Scientific Procedures Act) 1986, schedule 1 (appropriate methods of humane killing, http://www.legislation.gov.uk/ukpga/1986/14/schedule/1) procedure upon the owner’s request, following previously described protocols [14].

All snakes underwent a full postmortem examination. Samples from all major organs and tissues were collected for histological examination. These were fixed in 10% buffered formalin for appr. 24 h, trimmed and routinely embedded in paraffin wax. Sections (2–3 μm) were prepared and stained with haematoxylin and eosin (HE). Histological examination was performed on all internal organs, relevant diagnoses are compiled in Table S1. Spleen and/or thymus were examined in more detail, when available (spleen: n = 39; thymus: n = 24). In selected cases, consecutive sections were subjected to special stains to further characterize the components of the connective tissue in capsule and trabeculae: the Van Giesson stain served to highlight collagen fibers, the Reticulin stain for reticulin fibers, and the Resorcinfuchsin stain for elastic fibers, following previously published protocols [14]. To further characterize the “granular cells” observed in the lymphoid tissue and gather information on the composition of the cytoplasmic granules, we performed a series of special stains, including Prussian blue, Alcian blue, PAS, and Giemsa, following standard staining protocols.

BIBD was excluded in all animals by histological examination of HE stained sections of the internal organs (no evidence of the pathognomonic intracytoplasmic inclusion bodies in a wide variety of parenchymal cells) [18, 19]. For the majority of the animals, infection with reptarenaviruses, the causative agents of BIBD, had also been ruled out by multiplex PCR as part of another study [14]. The snakes included into the study were submitted for postmortem examination at various time points throughout the year (Table S1).

Morphometric analyses

Organ size

The spleen of B. constrictor is an ellipsoid organ [14]. Therefore, to allow a consistent approach to determine the actual organ dimensions, only spleens that had been bisected and longitudinally embedded were included into the study (n = 41). Both the long and short axes were measured using the Linear Measurement Tool in the NDP.view2 Image viewing software (Hamamatsu Photonics). The thymus was not dissected and embedded in a similar, consistent manner, as the organ was often difficult to fully delineate grossly; hence, to avoid inaccurate values, attempts at organ size measurements were abandoned.

For morphometric analyses, HE stained sections of spleen and thymus were scanned using a digital slide scanner (NanoZoomer-XR C12000; Hamamatsu, Hamamatsu City, Japan) and evaluated with the computer program VIS (Visiopharm Integrator System, Version 5.0.4. 1382; Visiopharm, Hoersholm, Denmark).

Organ cellularity

For the assessment of the overall cellularity of spleen and thymus, the scanned HE stained sections were assessed as previously described [14]. Briefly, the total area of lymphoid tissue in both organs (cortex and medulla of the thymus; and all lobules/intertrabecular regions of the spleen) was selected manually as a region of interest (ROI). Afterwards, a decision forest classification method was employed for the classification of cells and the results were presented as the total nuclei count per ROI area (in µm²).

Thickness of splenic and thymic capsule and trabeculae

The thickness of the capsule and trabeculae was measured in five representative locations of the spleen and thymus using the Linear Measurement Tool. The mean thickness was subsequently calculated for each, the capsule and the trabeculae.

Semiquantitative grading of selected histological features

Frequently occurring histological findings in spleen and thymus were assessed semiquantitatively to examine their potential relationship with age. The following grading system was applied:

Epithelial hyperplasia of the thymus: The thymic medulla generally exhibits some epithelial cells that form occasional small tubules. When these tubules were more frequent, epithelial hyperplasia was diagnosed, which was graded as mild (+), moderate (++) or severe (+++), with the latter grade, > 90% of the medulla was affected.

Granular cells in the spleen and the thymus: The extent of granular cell infiltration was assessed and graded: not observed (-); predominantly (multifocal) single cells and occasional small groups of 2–3 cells (+); moderate number of cells, predominantly in groups (++); and +++ abundant cells, found disseminated in or in close proximity to the connective tissue framework (+++).

Stromal fibrosis of the spleen: The extent of connective tissue deposition in the splenic lobules was assessed, in which (-) corresponded to not observed; + to a mild, ++ to a moderate and +++ to focally extensive to diffuse deposition, the latter spanning across two or more trabecules (+++).

Statistical analysis

All morphometric parameters and the grading of the histological findings were analysed using a GraphPad Prism software (Version 8.0.2., Boston, USA). The level of significance testing was set with a P value of 0.05. Descriptive statistics were applied, and the data were tested for normality by the D’Agostino-Pearson normality test. For the purpose of this study, the animals were categorized into three age groups: subadult, adult, and old. Since a former study had revealed significant differences in some blood parameters between boas pre- and post-sexual maturity and reproduction [20], we chose to apply the same cutoff to distinguish between subadult and adult boas (subadult referring to < 3 years of age; prior to definite sexual maturity and reproduction, and adult referring to ≥ 3 years of age; sexually mature, reproducing animals) to assess the lymphatic tissue-related changes. However, since the small sample size of the “old” boa group (n = 7) markedly limited the statistical power for meaningful comparisons of this group, for all statistical analyses we grouped the old boas together with adult boas (up to 15 years). Additionally, we also assessed differences between males and females to reveal a potential influence of sex on the examined parameters. Normally distributed data were analysed using two-sample t-test, and non-parametric tests (Wicoxon rank-sum/Mann-Whitney) were used for data that were not normally distributed. The arithmetic mean, including confidence intervals, was determined on normally distributed data and medians were reported where non-parametric tests were applied. A linear regression analysis was used to test if age significantly predicted changes in all morphometric parameters.

Results

Study population

The age of the animals included into the study ranged from 3 days to 21 years. Twenty-five were “subadult” (< 3 years of age), 17 “adult” (≥ 3–15 years of age; after sexual maturation) and 7 “old” (> 15 years). Twenty-one snakes were female, 14 male; for 13 juvenile animals, the sex could not be determined due to lack of differentiation of the gonads. Apart from one animal (No 15) that had shown intermittent dyspnea prior to death, the animals had not presented clinical signs prior to euthanasia or death. Pathological changes were rare and restricted to bilateral suppurative spectaculitis (animal No 12.3), acute vertebral fracture (animal No. 16.1) and mild granulomatous pneumonia (animal No 15.1). These conditions were not considered as likely to have affected the lymphatic tissue composition. Individual animal data are provided in Table S1.

Splenic size increases with age while the cellularity decreases

The length of the long and short axis served as a proxy for the size of the spleen. Both were significantly greater in the adult - old snakes (long axis: subadult snakes arithmetic mean = 2880 μm, Confidence interval (CI) = 2455–3304 μm, adult - old snakes arithmetic mean = 7372 μm, CI = 6745–8475 μm, with t = 78.309, df = 33, p < 0.0001, short axis: subadult snakes arithmetic mean = 2184 μm, CI = 1676–2693 μm, adult - old snakes arithmetic mean = 5904 μm, CI = 4837–7446 μm, with t = 6.238, df = 33, p < 0.0001) (Fig. S1A and B). The linear regression analysis revealed a significant positive correlation between age and splenic size, i.e. long and short axis (R² = 0.525, F = 38.63, p < 0.0001and R² = 0.503, F = 35.4, p < 0.0001, respectively), indicating progressive growth of the spleen with age (Fig. 1A). The detailed results are presented in Table S2A and B.

Fig. 1.

B. constrictor, spleen. Morphological and quantitative age-associated changes. (A) Splenic size. The size of the spleen (determined based on the length of both the long (green) and the short (blue) axis in HE stained cross sections) increases with advancing age, as shown by the positive correlation in the linear regression analysis (R² = 0.525, p < 0.0001 and R² = 0.503, p < 0.0001, respectively). (B) Splenic cellularity. The total number of the cells per lymphoid tissue area decreases with advancing age, as indicated by the negative correlation in the linear regression analysis (R² = 0.396, p < 0.0001). (C) Fibrous framework. The thickness of both the capsule and the trabeculae increases with advancing age, as indicated by the positive correlation in the linear regression analysis (R² = 0.799, p < 0.0001 and R² = 0.754, p < 0.0001, respectively). D-H. Morphological features of the spleen. D-G. Spleen of a subadult boa (animal 10.1; 3 months). (D) Cross section, showing densely cellular lymphatic tissue aggregates (asterisk) with thin fibrous capsule and trabeculae. HE stain, bar = 500 μm. E-G. Closer view of the fibrous framework, with a thin capsule and branching trabeculae (E: HE stain), mainly comprised of collagen fibers (F: Van Giesson stain, red fibers) with fewer embedded elastic fibers (G: Reticulin stain). Bars = 50 μm. H-K. Spleen of an old boa (animal 14.1; 16 years). H. Cross section, showing that the organ is larger than in the subadult snake and exhibits prominent capsule and trabeculae. HE stain, bar = 500 μm. I-K. Closer view of the fibrous framework, with a thick capsule and branching trabeculae (I: HE stain), mainly comprised of collagen fibers (J: Van Giesson stain) with fewer embedded elastic fibers (K: Reticulin stain (K). Bars = 50 μm

Comparison of the cellularity per µm² in the two age groups showed significantly higher values in the subadult snakes (subadult snakes median = 0.018/µm², CI = 0.018–0.019/µm², adult - old snakes median = 0.016/ µm², CI = 0.015–0.018, with z = 97, p < 0.001) (Fig.S1C). The linear regression analysis revealed a significant negative correlation between age and splenic cellularity (R² = 0.394, F = 25.31, < 0.0001), indicating that as age increases, splenic cellularity decreases (Table S2B and Fig. S1B). There was no significant difference in the splenic cellularity between female and male animals.

Thymic cellularity is lower in older, sexually mature Boas

For the thymus, comparison of the cellularity per µm² in the two age groups showed significantly higher values in the subadult snakes (subadult snakes median = 0.02/µm², CI = 0.019–0.027/µm², adult - old snakes median = 0.018/ µm², CI = 0.016–0.02, with z = 16, p < 0.01) (Table S2A and Fig. 2A). Although the linear regression analysis did not reveal a significant correlation between age and thymic cellularity, there was a trend for its reduction with age (Table S2B; Fig. 2B). There was no significant difference in the splenic cellularity between female and male animals.

Fig. 2.

B. constrictor, thymus. Morphological and quantitative age-associated changes A, B. Thymic cellularity. (A) Adult - old boas show significant fewer cells per lymphatic tissue area (cellularity) compared to subadult boas. Box and whisker plot, **p < 0.001. (B) The total number of cells per lymphatic tissue area shows a trend toward a decrease with advancing age, although no statistically significant correlation is observed in the linear regression analysis (R² = 0.17, p = 0.08). (C) Fibrous framework. The thickness of both the capsule and the trabeculae increases with advancing age, as indicated by the positive correlation in the linear regression analysis (R² = 0.33, p < 0.01 and R² = 0.43, p < 0.001, respectively). D-I. Morphological features of the thymus. D-F. Thymus of a subadult boa (animal 10.18, 3 months). Closer view of the fibrous framework, with a thin capsule (D: HE stain), mainly comprised of collagen fibers (E: Van Giesson stain, red fibers) with fewer embedded elastic fibers (F: Reticulin stain). Bars = 50 μm. G-I. Thymus of an old boa (animal 10.7, 20 years). Closer view of the fibrous framework, with a thick capsule and branching trabeculae (G: HE stain), mainly comprised of collagen fibers (H: Van Giesson stain) with fewer embedded elastic fibers (I: Reticulin stain (K). Bars = 50 μm

The fibrous framework of spleen and thymus increases with age

In the spleen, the thickness of both capsule and trabeculae was significantly higher in adult - old snakes than in subadult snakes (capsule: subadult snakes arithmetic mean = 44.08 μm, CI = 36.21–51.95 μm, adult - old snakes arithmetic mean = 177.6 μm CI = 122.1–224.8 μm, with t = 5.881, df = 37, p < 0.0001; trabeculae: subadult snakes median = 51.23 μm, CI = 46.5–65.3 μm, adult– old snakes median = 230 μm, CI = 167.7–277.3 μm, with z = 16, p < 0.0001) (Fig.S1D and E). The linear regression analysis revealed a significant positive correlation between age and both capsular and trabecular thickness (R² = 0.8, F = 151.5, p < < 0.0001 and R² = 0.754, F = 113.5, p < < 0.0001, respectively), indicating their progressive thickening with age (Table S2B; Fig. 1C-K). With the help of the different special stains it was shown that the observed thickening was mainly due to deposition of collagen fibers (making up approximately 90% of the splenic framework), interspersed with reticulin and elastin fibers. The proportion of the different connective tissue fibers did not appear to change with age (Fig. 1E-G; I-J).

The thymus of adult - old snakes also exhibited significantly thicker capsules and trabeculae (capsule: subadult snakes arithmetic mean = 12.85 μm, CI = 5.519–21.99 μm, adult - old snakes arithmetic mean = 72.2 μm, CI = 23.19–106.6 μm, with t = 4.845. df = 22. p < 0.0001 and trabeculae: subadult snakes arithmetic mean = 9.07 μm, CI = 6.483–13.82 μm, adult -old snakes arithmetic mean = 32.05 μm, CI = 18.76–44.89 μm, with t = 6.792. df = 21. p < 0.0001) (Fig. S2A and B). The linear regression analysis revealed a significant positive correlation between age and the capsular and trabecular thickness (R² = 0.381, F = 13.56, p < 0.05 and R² = 0.564, F = 27.22, p < 0.0001, respectively), indicating their progressive thickening with age (Table S2B; Fig. 2C). The composition of the framework was similar to the one of the spleen and did not change with increasing age (Fig. 2D-I). No significant differences between the two sexes were noted in regard to the splenic or thymic framework.

Histopathological changes in spleen and thymus and their potential association with age

We have recently described the basic architecture and cell composition of spleen and thymus in B. constrictor in detail [14]. While this study also reported any histopathological changes, their potential association with age was only marginally discussed. Here, we report several histological features in spleen and thymus that appear to be more frequent and rather consistent in older animals. Table S1B provides a list of the findings and includes a semiquantitative grading of their extent. Additionally, Table S3 presents a narrative summary of histological findings in the three age classes, i.e. subadult, adult and old snakes.

Granular cell infiltration

In a previous study, we have identified the so-called granular cells (characterized by abundant yellowish to orange-colored granules in their cytoplasm) in thymus and spleen of boa constrictors where they were particularly evident in animals with BIBD. Although we could not determine the exact origin of these cells, we concluded that they are likely leukocytes, which may be present in higher numbers in association with chronic inflammatory disease processes [14]. Here, we found predominantly subcapsular aggregates of granular cells in both spleen and thymus (Fig. 3A and B) of most examined animals, and across all age groups (28/41 in the spleen (68.3%), 12/25 in the thymus (48%); age range of affected animals: 3 days to 21 years). The grade of granular cell infiltration was significantly higher in both organs in adult - old boas compared to subadult boas, with z = 15,50, p < 0.0001 for the spleen and z = 2, p < 0.05 for the thymus (Figs. S1F and 2 C, respectively). The highest grade was observed in the four oldest boas, aged 20 and 21 years. The panel of special stains applied to characterize the granular cells further did not yield clear results: the granules did not consist of iron (Prussian blue stain), mucopolysaccharides and glycosaminoglycans (mucin etc., Alcian blue stain) or polysaccharides (glycogen etc., PAS reaction) (Fig.S3A-C). The vast majority turned pinkish to reddish when subjected to the Giemsa stain (Fig.S3D).

Fig. 3.

Age-related findings in the spleen and thymus of B. constrictor. A, B. Spleen, granular cell infiltration (animal 10.7; 20 years, severe degree). (A) Granular cell aggregate in the subcapsular region (arrow). (B) Closer view of granular cells found in groups (arrowhead) or as single cells (arrow). C, D. Spleen, stromal fibrosis (animal 10.14; 21 years, severe degree). Connective tissue (collagen fibres) replaces the lymphatic tissue (asterisks). D. Closer view of a collagen fiber deposition (asterisk). Note also the presence of individual granular cells (arrow and inset). E, F. Thymus, epithelial cell hyperplasia (animal 10.20; 18 years, moderate degree). E. Epithelial cells form tubular structures in the thymic medulla and cortex (arrow). F. Closer view of the variably sized tubular structures lined by epithelial cells. The lumen contains homogenous eosinophilic (proteinaceous) material (arrow). HE stains, bars = 50 μm. Inset: HE stain, bar = 10 μm

Stromal fibrosis in the spleen

In 12/22 (54.5%) adult - old boas (10–21 years), the spleen exhibited multifocal to coalescing deposits of connective tissue (collagen fibers and fibrocytes) of mild to severe degree; these extended from capsule or trabeculae into the parenchyma and partially replaced the splenic lobules (Fig. 3C and D). The highest grade was observed in the four oldest boas, aged 20 and 21 years. It is likely that this feature is linked to the progressive thickening of the capsule and trabeculae that is seen with increasing age.

Epithelial hyperplasia in the thymic medulla

We have previously reported epithelial hyperplasia in the thymus in adult - old snakes, characterized by an increased number of cuboidal to columnar epithelial cells within the thymic medulla [11]. We also observed epithelial hyperplasia in the thymus in the current cohort, in the adult - old snakes (8/11, 72.7%; age range: 5 to 20 years); it was not present in the thymus of subadult snakes (n = 14). The extent of epithelial hyperplasia varied overall, with both “younger” adult animals (aged 5 to 6 years) and old snakes (20 years) exhibiting mild, moderate, and severe degrees. The epithelial cells formed small ductal structures that were occasionally slightly dilated (Fig. 3E and F). The higher occurrence in adult snakes suggests the feature as a condition potentially linked to sexual maturity and age.

Capsular adipocyte accumulation

In two animals (Nos 10.15 and 12.2, aged 11 and 21 years; 5.1%), multiple small aggregates of mature adipocytes (fat tissue) were present between the collagen fibers of the splenic capsule (Fig. 4A). Similar aggregates were observed in the thymic capsule of two further animals (Nos 3 and 10.6, aged 4 and 6 years, 8.3%). Given the varying ages of the affected snakes, this likely represents an incidental change.

Fig. 4.

Incidental findings in the spleen and thymus of B. constrictor. A. Thymus, intracapsular adipocytes (animal 3.1; 4 years). Accumulations of mature adipocytes (asterisks) in the thymic capsule. B. Spleen, nodule formation (animal 10.8; 5 years). A nodular accumulation of lymphatic tissue extends from the capsule. C, D. Thymus, cyst (animal 10.6; 6 years). (C) Overview, showing a large cavity filled with amorphous pale eosinophilic homogenous to granular material (asterisk), demarcated by a fibrous capsule and lymphatic tissue (arrow). (D) The cyst is lined by a columnar epithelium consisting of goblet cells (arrowhead). Note also the fibrous capsule and infiltrating granular cells (arrow). HE stains, A, B, D: bars = 50 μm, C: bar = 500 μm

Splenic nodules

The spleen of 5 animals (Nos. 5.1, 5.2, 10.4, 10.5, 10.19; aged 3 days to 5 years, 12.8%), exhibited singular, well demarcated nodules of splenic lymphatic tissue protruding from the capsule (Fig. 4B). The nodules were covered by a thin rim of fibrous tissue. Similar to the intracapsular adipocyte accumulations, due to the broad age range of the animals affected, this finding also was considered an incidental lesion.

Thymic cysts

In three snakes (Nos 10.19, 10.6, 10.16, 10.19; aged 3 months, 6 and 10 years, 12.5%), the thymus exhibited large cystic cavities (200 μm to 2 mm in diameter) lined by a monolayered cuboidal to columnal epithelium with frequent apical ciliation and mucus globules in the cytoplasm (Fig. 4C and D). Their lumen contained amorphous pale eosinophilic homogenous to granular (proteinaceous) material, eosinophilic crystals and/or cell debris. Due to its occurrence across a broad age span, this change is interpreted as an incidental finding.

Discussion

In humans, aging of the lymphatic organs is associated with distinct structural and functional alterations that collectively contribute to the progressive decline in immune function observed with advancing age (also termed immunosenescence) [21–23]. Studies in humans have also shown that, on a cellular level, immunosenescence is linked to changes in immune cell populations, such as a reduction in the number of naive T cells and B cells, loss of memory cells, impaired antigen presentation, reduced T and B cell receptor repertoire diversity, or accumulation of senescent cells [2, 21, 24]. These alterations can impair the formation of immunological memory and the ability of an organism to respond to new antigens and therefore lead to increased susceptibility to disease [1, 21]. The investigation of the mechanisms behind immunosenescence across various animal taxa, and in particular among lower vertebrates such as reptiles, is significantly constrained by the limited knowledge on the morphology and function of their immune cells and immunological processes [25, 26]. Specifically, fundamental components of adaptive immunity, such as naive and memory T and B cells, the structural and functional characterization of T cell and B cell receptors, and the presence and roles of natural killer cells are poorly described or have not even been identified in the order Reptilia [25, 27]. The present study took an initial step towards identification of the effect of aging on the immune system of the Boa constrictor, a boid snake species frequently kept and bred in captivity, by describing the morphology of age-related changes in the lymphoid tissue in this species. This also served to determine whether there is evidence of immunosenescence in the species (and reptiles/poikilothermic animals) in general.

Our study revealed a significant progressive decrease in lymphoid cellularity in the spleen with age, while the size of the organ, as determined by measuring its long and short axes, increased significantly. These findings are consistent with studies in laboratory mice, in which the spleen’s weight increased with advancing age, yet exhibited reduced cellularity, primarily attributed to a decrease in lymphocyte numbers [20]. Aging in mice was also associated with further splenic alterations, such as an increase in macrophages, a rearrangement of the microenvironment (in particular an increase in reticular cells), and the loss of well-defined follicular structures [20]. The assessment of such features and hence a comparative approach is not possible in the B. constrictor as the boa spleen lacks a clear, organized structure. To specify, the spleen is entirely devoid of lymphoid follicles (i.e. no evidence of germinal centres, marginal zone, and mantle zone, as typically observed in mammals [21]) and a red pulp equivalent, as we described in an earlier study [14]. Furthermore, while T cells and macrophages can be identified by immunohistochemistry with cross reacting antibodies, there seem to be only few, potentially randomly distributed B cells (as shown by RNA-ISH for CD20 mRNA) and hardly any plasma cells (so far, we only found ultrastructural evidence of their existence in boas) [14]. Given in particular the lack of reliable immunohistochemical markers for all relevant reptilian leukocyte types, we decided to not pursue the assessment of potential age-associated changes in the composition of the lymphoid tissue any further for the current study.

In our study, both lymphoid organs exhibited significant alterations in the connective tissue fibre framework, characterized by a progressive thickening of capsule and trabeculae as the boas aged. Although this phenomenon has not been extensively studied across animal species, it is well documented in human medicine, where older individuals show an overall increase in the thickness of the splenic capsule [2, 28–30]. Interestingly, these studies have indicated that the apparent thickening is primarily due to an increase in collagen fibres, while other connective tissue components, such as reticulin and elastin fibres, decrease in number [30]. The progressive reduction in elastin fibres in the splenic capsule has been suggested to limit splenic distention and contribute to its involution with aging in humans [30]. The present study found no evidence of any significant differences in the proportion of connective tissue components with the fibrous thickening of capsule and trabeculae in spleen and thymus. These findings suggest progressive extracellular matrix (ECM) synthesis and deposition in boas with age but no ECM remodelling and hence possibly a different underlying mechanism compared to humans.

One of the most consistent histological findings associated with advancing age in B. constrictor was the presence and number of so-called granular cells in both thymus and spleen. So far, the origin of this cell type is not known. Granular cells do not express known macrophage (such as Iba-1) or lymphocyte markers [14]. Application of a panel of special stains showed that they do not contain iron, mucin or polysaccharides (glycogen etc.). Interestingly, the cytoplasmic granules turned bright pink with the Giemsa stain. However, this staining reaction does not suggest a specific composition of the granules, as it does not correspond to the characteristic metachromatic response of mast cell granules, that is well documented in mammals and appears to also apply to mast cells in the boa constrictor [31, 32]. Given these findings, it appears unlikely that the granular cells observed represent mast cells in Boa constrictor. Eosinophils, in principle another option, have so far only been described in blood smears but not in histological specimens of boas [20]. Considering that the staining properties of reptilian leukocytes appears to differ from that of most mammals (i.e. “heterophils” instead of “neutrophils”), we cannot differentiate the granular cells further at this stage but feel confident that they are indeed leukocytes. In any case, the apparent increasing occurrence of these presumable leukocytes in the lymphoid organs is noteworthy considering that “inflammageing” is a key component of immunosenescence in human ageing. The term refers to a state of chronic, low-grade inflammation that tends to intensify with age and is characterized by an increased infiltration of organs with inflammatory cells [21, 24]. Especially macrophages seem to play a key role for induction and maintenance of inflammageing, as their dysfunction during ageing reduces their capacity to remove senescent cells from tissues [21, 24]. It is worth mentioning that in humans, inflammageing has also been shown to promote the activation of fibroblasts and other ECM-producing cells, leading to the excessive deposition of ECM proteins and fibrosis in internal organs [21]. In this regard, the high number of granular cells in a predominantly subcapsular localization in both thymus and spleen in older boas hints towards an association between the infiltration with granular cells and the observed progressive fibrosis.

The present study confirmed an overall significant decrease in thymic cellularity in adult compared to subadult boas; however, linear regression analysis only showed a trend towards a negative correlation between thymic cellularity and age, it did not reach statistical significance. Since we could identify the thymus as a distinct organ also in aged snakes, up to 21 years of age, after many years of sexual maturity, these findings might suggest that the “classical” thymic involution, as observed in mammals and birds [7, 33–36], does not take place in this snake species. The reason for this phenomenon remains unclear. However, given that the lymphoid organs of B. constrictor are predominantly composed of T cells, as outlined in our previous study [14], it can be speculated that the thymus plays an essential in T cell development, selection and maturation through a boa’s lifetime.

We also observed other histological changes in the lymphatic tissue of older boas, such as epithelial hyperplasia in the thymic medulla. We have already described this feature that is common in association with thymic involution in mammals [34–36] in B. constrictor [14]. In contrast to epithelial hyperplasia, thymic cysts, which are also frequently linked to thymic involution in mammals, were occasionally observed across all age groups in the current study, providing further evidence that “classical” thymic involution does not occur in this snake species. The unilocular, thin-walled nature of these cysts, along with the presence of ciliated epithelium, suggests a congenital nature; indeed, they might arise from remnants of the brachial arch epithelium [37].

Conclusions

In summary, this study provides first evidence of age-associated changes in the immune system of Boa constrictor, a species that, like reptiles in general, has so far received very limited attention in the context of immune aging. Given the large number of species within the class Reptilia (more than 12000 species are known today [38]), their high biological and life span variability requires reframing our understanding of immunosenescence. However, our study indicates that certain morphological alterations, such as reduced cellularity, progressive fibrosis, and low-grade inflammation, are present and appear to parallel those observed in humans and in other animal classes. Future research is needed to determine the extent to which these morphological changes are associated with functional impairments at cellular and/or organ level, in order to allow a more definitive statement regarding the occurrence of immunosenescence in snakes. Additionally, the development of more tools to identify and assess the function of leukocyte subtypes in reptiles, including the so-called granular cells, would allow further investigations into the mechanisms of inflammaging and progressive fibrosis in aging reptiles.

Electronic supplementary material

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Author contributions

E.D.: Conceptualization, methodology, investigation, writing (original draft, review & editing). U.H.: Writing (review& editing). A.K.: Conceptualization, writing (review & editing), supervision.

Funding

This work was conducted without external funding.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All procedures in this study were approved by the institutional review board (MeF-Ethik-2024-01). The terms of service to which owners agree when submitting an animal for a diagnostic post mortem examination include the permission to make use of material from the examination for both teaching and research. An animal experiment license is not required for conducting examinations on diagnostic material.

Consent for publication

All authors have approved the paper for publication.

Competing interests

The authors declare no competing interests.