Plasticity of Schwann cells and pericytes in response to islet injury in mice
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Islet Schwann (glial) cells and pericytes are the microorgan’s accessory cells positioned at the external and internal boundaries facing the exocrine pancreas and endothelium, respectively, adjacent to the endocrine cells. Plasticity of glial cells and pericytes is shown in the glial scar formation after injury to the central nervous system. It remains unclear whether similar reactive cellular responses occur in insulitis. We applied three-dimensional (3D) histology to perform qualitative and quantitative analyses of the islet Schwann cell network and pericytes in normal, streptozotocin-injected (positive control of gliosis) and NOD mouse models.
Vessel painting paired with immunostaining of mouse pancreatic tissue was used to reveal the islet Schwann cells and pericytes and their association with vasculature. Transparent islet specimens were prepared by optical clearing to facilitate 3D confocal microscopy for panoramic visualisation of the tissue networks.
In-depth microscopy showed that the islet Schwann cell network extends from the peri-islet domain into the core. One week after streptozotocin injection, we observed intra-islet perivascular gliosis and an increase in pericyte density. In early/moderate insulitis in the NOD mice, perilesional gliosis occurred at the front of the lymphocytic infiltration with atypical islet Schwann cell morphologies, including excessive branching and perivascular gliosis. Meanwhile, pericytes aggregated on the walls of the feeding arteriole at the peri- and intralesional domains with a marked increase in surface marker density.
The reactive cellular responses demonstrate plasticity and suggest a stop-gap mechanism consisting of the Schwann cells and pericytes in association with the islet lesion and vasculature when injury occurs.
KeywordsGliosis Insulitis Islet NOD mouse Pericyte Plasticity Schwann cell Streptozotocin
Central nervous system
Glial fibrillary acidic protein
Neuron-glial antigen 2
The endocrine islets of Langerhans are the regional neurovascular hubs in the pancreas and constantly receive signals from the circulation and nerves in response to physiological cues . Associated with the rich nerve and blood supplies, islets are sheathed in a dense network of the processes of Schwann cells (the glial cells of the peripheral nervous system) [2, 3] and are positioned with pericytes along the capillaries [4, 5]. However, due to the dispersed nature of the neural and vascular tissues, characterisation of the islet Schwann cells and pericytes in health and disease has been difficult, even in animals.
Notably, in the central nervous system (CNS), gliosis and glial scar formation, consisting of the glial cells and pericytes surrounding the lesion, are the hallmarks of Alzheimer’s disease, stroke and traumatic injury [6, 7, 8]. In the enteric nervous system, increases in the expression of glial fibrillary acidic protein (GFAP) and proliferation of glial cells have also been reported in bowel inflammation such as ulcerative colitis [9, 10, 11, 12]. As to the ‘neuroendocrine’ islet, it remains unclear whether similar reactive cellular responses occur in islet injuries, such as insulitis, in the progression of type 1 diabetes.
Using NOD mice, which have a condition resembling type 1 diabetes with insulitis caused by lymphocytic infiltration, Winer et al discovered that the autoimmune target tissues include both pancreatic beta cells and Schwann cells . They found that the peri-islet lymphocytic accumulation in insulitis creates disturbances and breaches of the glial sheath prior to the autoimmune attack on the beta cells [14, 15, 16]. Interestingly, in contrast to the destruction of Schwann cells, reactive gliosis was also reported in streptozotocin (STZ)-injected mice . The variation in islet glial-cell content in animal models of diabetes highlights Schwann cells as a dynamic cellular factor involved in islet injury and inflammation.
In rodent models of obesity and hypertension, histological changes in islet pericytes, including hypertrophy and an increase in staining density of the surface marker neuron-glial antigen 2 (NG2), have been observed [4, 5, 18, 19], suggesting pericytes’ morphological plasticity in response to the disturbances of the circulation. In the progression of type 1 diabetes, although the role of pericytes is not understood, vascular swelling and leakage have been imaged in insulitis and used as an indicator of islet inflammation in NOD mice [20, 21]. Whether or not the vascular damage induces remodelling of pericytes is the topic for investigation in this research.
To elucidate the activity of Schwann cells and pericytes in islet injury, we prepared transparent islet specimens by optical clearing [22, 23, 24, 25] and combined vessel painting (perfusion of fluorescent lectin) with three-dimensional (3D) histology [26, 27, 28, 29, 30, 31, 32]. This allowed the identification of the spatial features of the GFAP+ Schwann cells and NG2+ pericytes (note: although named as a neuron-‘glial’ antigen, the NG2 cell-surface chondroitin sulfate proteoglycan is highly expressed in the pancreatic/islet pericytes and has been used as a histological marker [5, 33]) and their association with the islet lesion and vasculature [13, 17, 34, 35, 36]. Qualitative and quantitative analyses of the islet injury-induced gliosis and pericyte remodelling around the feeding arteriole, and their pathophysiological implications, are presented and discussed in this report.
Pancreases harvested from female BALB/c mice (BioLASCO, Taipei, Taiwan) were used to acquire the images of normal islets. Pancreases harvested from streptozotocin (Sigma, St Louis, MO, USA)-injected female BALB/c mice (age 7–8 weeks; single i.p. injection, 180 μg/g body weight; killed at one week after the injection, positive control of gliosis ) and female NOD mice (age 7–8, 11–14 and 20–22 weeks; National Laboratory Animal Center, Taipei, Taiwan) were used to acquire images of different scales of insulitis. Schwann cell destruction in severe insulitis was used as the negative control of GFAP staining.
Islet injury induced by STZ injection was confirmed by hyperglycaemia (6 h fasting glucose concentration >8.3 mmol/l) measured on day 3 (from the tail tip with an Accu-Check Performa glucometer; Roche, Indianapolis, IN, USA) and day 7 before mice were killed (six out of eight STZ-injected mice developed hyperglycaemia; only the mice with hyperglycaemia were used in microscopy and analysis).
The glucose levels of the untreated BALB/c and NOD mice in early and moderate insulitis were within the normal range (3.9–6.7 mmol/l). The young NOD mice were used to study the islet lesions and perilesional tissue remodelling induced by autoimmune progression. The aged NOD mice with severe insulitis and hyperglycaemia were used as a negative control to reveal the destruction of the Schwann cell network, as previously shown by Winer et al . Overall, 44, 30 and 36 image stacks derived from six normal BALB/c mice, six STZ-injected BALB/c mice and eight NOD mice (three at age 7–8 weeks, three at age 11–14 weeks and two at age 20–22 weeks), respectively, were used to generate representative images. The National Tsing Hua University Institutional Animal Care and Use Committee approved all procedures involving mice.
Vessel painting [30, 34, 35] was performed by cardiac perfusion of the lectin (wheat germ agglutinin)–Alexa Fluor 488 conjugate (30 μg/g body weight; Cat. No. W11261; Invitrogen, Carlsbad, CA, USA) (pseudo-coloured red was assigned to the signals of blood vessels in the images) followed by 4% paraformaldehyde perfusion fixation. After this, pancreases were harvested and the vibratome sections of the tissue (∼ 400 μm) were post-fixed in 4% paraformaldehyde solution for 1 h at 25°C. The fixed tissues were then immersed in 2% Triton X-100 solution for 2 h at 25°C for permeabilisation.
The primary antibodies used to reveal the islet Schwann cells and pericytes were a polyclonal rabbit anti-GFAP antibody (DAKO, Z0334; Carpinteria, CA, USA) and a rabbit anti-NG2 antibody (AB5320; Millipore, Billerica, MA, USA), respectively. Before applying the antibody, the tissue was rinsed in PBS. This was followed by a blocking step, in which the tissue was incubated with a blocking buffer (2% Triton X-100, 10% normal goat serum and 0.02% sodium azide in PBS). The primary antibody was then diluted with a dilution buffer (1:50; 0.25% Triton X-100, 1% normal goat serum and 0.02% sodium azide in PBS) to replace the blocking buffer and incubated for 1 day at 15°C.
The Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (1:200; Invitrogen) was used to reveal the immunostained structure. Then nuclear staining by propidium iodide (50 μg/ml; Invitrogen) was performed at room temperature for 1 h. The labelled specimens were then immersed in the optical-clearing solution FocusClear (CelExplorer, Hsinchu, Taiwan) overnight before being imaged via confocal microscopy ).
A Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) equipped with the objectives of 20× (optical section: 5 μm; Z-axis increment: 2.5 μm) ‘Fluar’ lenses and 40× (optical section: 3 μm; Z-axis increment: 1.5 μm) LD ‘C-Apochromat’ water immersion lenses (working distance: 620 μm) was used to acquire the images. We used transmitted light microscopy in combination with confocal microscopy to examine the specimens. Each micrograph consisted of 1,024 (X) × 1,024 (Y) pixels. The laser-scanning process was operated under the multi-track scanning mode to sequentially acquire signals in multiple channels. The Alexa Fluor 647-labelled structures were excited at 633 nm and the fluorescence was collected by the 650–710 nm band-pass filter. The propidium iodide-labelled nuclei were excited at 543 nm and the signals were collected by the 560–615 nm band-pass filter. The lectin–Alexa Fluor 488-labelled blood vessels were excited at 488 nm and the fluorescence was collected by the 500–550 nm band-pass filter.
Image projection and analysis
Quantification of pericytes in the normal pancreas (Fig. 2d) and the STZ-treated islets (Fig. 4c) was performed by counting the pericytes in the segmented volume of interest (endocrine islet or exocrine acini) for estimation of the cell densities. While counting the pericytes, both the NG2 and nuclear signals were used to define a pericyte—an NG2 immunoreactive cell body with at least two processes contacting the blood vessels. The ‘Label Field’ function of Avizo was used to label and measure the endocrine and exocrine tissue volumes. For quantification of the pericyte remodelling on the feeding arteriole (Fig. 6f), we first chose and segmented the voxels of the arteriole, such as the blood vessels in the squashed circle in Fig. 6e. We then calculated the NG2 staining density by dividing the overall NG2 signals in the selected area by the voxels of the arteriole in the 3D image stack.
Four normal and four STZ-injected BALB/c mice (12 image stacks each) were used to estimate the increase in GFAP signals in the islets after STZ injection. Three normal and four STZ-injected BALB/c mice were used to estimate the pericyte density in the normal pancreas (13 and 12 image stacks of endocrine islets and exocrine acini, respectively) and in the STZ-treated islets (15 image stacks), respectively. Three normal BALB/c mice (8 image stacks) and five NOD mice in early/moderate insulitis (12 image stacks) were used to estimate the NG2 staining density in the blood vessels. Signal or pericyte densities derived from different image stacks of the same mouse were first normalised and then averaged over the other mice in the same group.
The quantitative values are presented as means ± SD. Statistical differences were determined by the unpaired Student’s t test. Differences between groups were considered statistically significant when p < 0.05.
In-depth characterisation of the peri- and intra-islet Schwann cell network
Owing to the dispersed nature of the cellular processes, the standard microtome-based tissue analysis cannot provide an integral view of the islet Schwann cell network. To increase the imaging depth, we prepared transparent islet specimens by optical clearing , which allowed us to use transmitted light microscopy to identify the major pancreatic components (the endocrine islet, pancreatic duct and exocrine acini) and visualise the peri- and intra-islet Schwann cell fibres via deep-tissue confocal microscopy (Fig. 1a–c and ESM Video 1 [first half]).
Three features of the Schwann cell network were seen in the gross image of islets in the pancreas. First, we observed minimal, if any, Schwann cell fibres associated with the acini in comparison with the condensed islet Schwann cell sheath and the slender fibres around the duct. Second, the Schwann cell plexus entered from the exocrine domain with multiple entry points into the islet mantle, forming a condensed fibre sheath. Third, we made an unexpected discovery, that the intra-islet Schwann cell fibres extended from the peri-islet domain into the core.
Using the transparent specimen, we next zoomed in to investigate the intra-islet Schwann cell fibres. Both in-depth observation and projections (Fig. 1d–f and ESM Video 1 [second half]) confirmed that the Schwann cell plexus sprouted from the sheath with processes extending into the core. In addition, the paths of the Schwann cell extensions were, in part, adjacent to those of the microvessels, implying their intimate association. The elongated Schwann cell processes from the mantle into the core suggested a previously overlooked role of the network, likely that it senses/monitors the intra-islet microenvironment, in addition to sheathing the islet.
To verify the endocrine islet structure, paired immunostaining of glucagon with GFAP was used to reveal the peri-islet alpha cells and the Schwann cell sheath (ESM Fig. 1). The glucagon-expressing alpha cells were used as a reference to define the mantle of the mouse islet, in which the peri-islet Schwann cell network and alpha cells were in contact with each other and in close proximity, confirming the location of the GFAP immunostaining signals.
3D Characterisation of islet pericytes
Similarly to the Schwann cells, pericytes with their long processes in space cannot be easily portrayed by standard two-dimensional (2D) microscopy. We used the developed penetrative imaging method with NG2 staining to characterise the 3D features of the pericytes. Figure 2a–c, ESM Fig. 2 and ESM Video 2 present three examples of the immunolabelled pancreatic/islet pericyte population associated with the vasculature (normal BALB/c). As can be seen, the NG2+ pericytes consisted of a cell body with a prominent nucleus (yellow arrows in Fig. 2a) and processes extending from the cell body to embrace the abluminal side of the vessel wall. This morphology was consistent with the 2D pericyte images shown in the literature [5, 33].
Importantly, through in-depth projection of pericytes with blood vessels, two additional features of the pancreatic/islet pericytes were seen. First, we observed the NG2+ processes/fibres encircling the arterioles (white arrows in Fig. 2a,b) in comparison with the extended NG2+ processes on the capillaries. Second, quantification of pericytes showed that the pericyte density was approximately fourfold higher in the endocrine islet than in the exocrine acini (Fig. 2c,d) (note: we quantified the pericytes as the NG2+ cells with at least two processes extending from the cell body in association with the blood vessels). The higher pericyte density in the islet reflected its rich vascularisation.
Plasticity of Schwann cells and pericytes in response to STZ-induced islet injury
Teitelman et al reported that STZ-induced islet injury stimulates neurotrophin expression and reactive gliosis of the Schwann cells . Because STZ injection also causes islet microvascular damage [38, 39, 40, 41], we sought to investigate the remodelling of Schwann cells and pericytes around the blood vessels after STZ injection (Fig. 3).
Figures 3a–d and ESM Video 3 show a middle-sized islet (∼150 μm) with reactive gliosis one week after the STZ injection. Merged projection of GFAP and capillaries revealed intra-islet perivascular gliosis, highlighted by the abundant and dispersed Schwann cell fibres contacting and docking on the capillary walls from different directions in the islet core. This is different from the elongation of the fibres following the paths of the capillaries seen in the normal islet (Fig. 1d–f). The induced intra-islet perivascular gliosis suggests that the Schwann cells not only form a sheath to separate the islet from acini but also sense the change inside the islet in response to injury.
Quantification of the GFAP signals showed that in the islet core, the density of the GFAP+ Schwann cell fibres was significantly higher than the normal density following the STZ injection (3.9-fold increase, Fig. 3e). In the mantle, however, because the islets had already been sheathed with a dense network of the processes of Schwann cells, there was no statistical difference between the normal and diseased condition.
For the pericytes, we used high-resolution 3D microscopy to detect the changes following STZ injection. Morphological analysis (Fig. 4) revealed atypical lateral spreading of the pericyte processes (yellow arrows in Fig. 4b) compared with the slender, longitudinal extensions of the normal processes on the capillary walls (Fig. 4a). Quantitative analysis (Fig. 4c) showed a modest 66% increase in pericyte density in the STZ-treated islet compared with the untreated control.
Lymphocytic infiltration induces localised gliosis in early and moderate insulitis in NOD mice
Unlike the generalised intra-islet injury induced by STZ injection, the lymphocytic accumulation in early insulitis in NOD mice caused localised islet injury (Fig. 5). The hallmark of the inflammatory response was the accumulation of lymphocytes between the duct and the islet . The lymphocytic invasion not only damaged the vasculature but also induced gliosis surrounding the influenced area (Fig. 5a–c).
Figure 5d–f shows the close-up view of the localised gliosis in early insulitis. Taking advantage of the transparent specimen, we acquired in-depth images of Schwann cells to demonstrate their close association with the infiltrated lymphocytes (ESM Video 4 [first half]). Zoom-in examination and projections of the Schwann cell processes and capillaries in Fig. 5e,f revealed that the induced gliosis occurred at the front of the infiltrated area against the normal domain (perilesional gliosis) with abundant short extensions sprouting from the Schwann cell sheath to contact the islet capillaries from various directions (perivascular gliosis). This morphology differed from the normal smooth and elongated Schwann cell fibres extending from the mantle into the core (Fig. 1d,f).
In moderate insulitis the perivascular gliosis extended from the mantle to the core together with the advancement of lymphocytic infiltration (Fig. 5g–i). High-resolution images at the infiltration boundary revealed perivascular gliosis with excessive branching of the Schwann cell processes on the capillary walls (Fig. 5i and ESM Video 4 [last part]). ESM Fig. 3 shows a second example of an islet in moderate insulitis, in which perivascular gliosis and the Schwann cell breach were observed simultaneously at different corners of the islet. The breach underlined the breakdown of the Schwann cell network due to the autoimmune attack as reported by Winer et al .
In severe insulitis, destruction of the Schwann cell network became prominent in the NOD mice (Fig. 5j–l). Under the intensive autoimmune attack, islet destruction was accompanied by vascular damage and dismantling of the Schwann cell network. Nonetheless, prior to the destruction, the transitory and localised gliosis and its association with the lesion indicate the plasticity of the islet Schwann cells and their participation in the inflammatory response in insulitis.
Pericyte aggregation around the feeding arteriole in insulitis in NOD mice
In addition to the perilesional gliosis, the lymphocytic infiltration also led to changes of pericytes around the islet feeding arteriole in the NOD mice. Unlike the STZ-induced global change of islet pericytes on the capillaries, lymphocytic infiltration primarily induced localised pericyte aggregation around the feeding arteriole at locations close to the islet pole. This phenomenon occurred while the lymphocytes arrived at the peri-islet domain (Fig. 6a,b) and persisted as the lymphocytes migrated into the core (Fig. 6c–e). The remodelling of pericytes on the walls of arterioles was distinctly observed in projection. We analysed 12 islets derived from five NOD mice in early/moderate insulitis to confirm this remodelling of pericytes on the walls of arterioles. The marked increase (4.7-fold) in the NG2 staining density on the arteriole was quantified (Fig. 6f) by collecting the signals of the NG2+ processes enclosing the blood vessel against those of the slender, perivascular NG2+ processes. The morphological and quantitative changes in pericytes on the feeding arteriole, as well as the perilesional and perivascular gliosis of Schwann cells in lymphocytic infiltration, highlight the plasticity of the two cell types in the progression of autoimmune diabetes.
The role of Schwann cells and pericytes in normal islet function is not completely understood, but their changes in response to islet injury indicated that they are not inert bystanders in experimental diabetes. Instead, they reacted to STZ injection and autoimmune progression in mouse models of diabetes. In particular, the Schwann cells responded to lymphocytic infiltration with gliosis occurring at the boundary of the islet lesion in early and moderate insulitis (perilesional gliosis, Fig. 5a–i). Gliosis was also found around the islet capillaries as the lesion progressed from the mantle into the core (perivascular gliosis, Fig. 5d–i). In the meantime, changes in pericytes occurred on the islet feeding arteriole at the peri- and intralesional regions (Fig. 6). Aggregation of pericytes, with their processes enclosing the vessel wall, underlined the pericytes’ plasticity. The reactive cellular responses demonstrate the plasticity and suggest a stop-gap mechanism consisting of the Schwann cells and pericytes in association with the islet lesion and vasculature while injury occurs in the autoimmune progression.
Studies on scar tissue in CNS injures have inspired this investigation into the Schwann cells and pericytes in islet injury. The formation of glial scar (including a stromal component derived from pericytes ) in CNS injuries is thought to help restore the blood–brain barrier. Here, in the progression of the islet lesion in the NOD mice, the Schwann cells and pericytes also targeted blood vessels in their reactive cellular responses, albeit without forming a scar. Nonetheless, the reactive cellular responses underline the critical role of the islet circulation in its endocrine functions.
In the NOD mice, we demonstrated that perilesional gliosis occurs in early and moderate insulitis before the destruction of the Schwann cells in severe insulitis. Using the same mouse model, Winer and colleagues showed that the accumulation of T cells around the islet first disrupts the Schwann cell network to create breaches before the attack on the beta cells [13, 43]; however, their study did not identify the phase of gliosis or the formation of the glial–vascular complex in the autoimmune progression. Based on our data shown in Fig. 5 and ESM Video 4, we propose that the Schwann cell disruption includes two phases: first, gliosis, which associates with the lesion and amplifies the antigenic/inflammatory signals (due to the antigen presenting ability of the Schwann cells [44, 45]) and, second, destruction, as a consequence of the lymphocytic attack.
Although we identified two new cellular responses that occurred in the islet lesion progression, we did not study their interactions. This is because the current tissue labelling and confocal imaging designs were limited to three fluorescence channels, which we felt confident avoided signal crosstalk, to present the image data with high fidelity (note: the ultraviolet channel was not used in this research due to limited penetration depth). Because two of the three channels were designated for observing the lesion (nuclear staining) and vasculature (vessel painting), only one was left to identify the marker of interest, either GFAP or NG2. Thus, we chose to characterise the Schwann cells and pericytes individually, but did not rule out their potential interaction or association with other network elements such as the nerves in the perilesional region. In fact, we used the same mouse model and imaging approach to reveal the perilesional remodelling of sympathetic nerves in insulitis , suggesting the generic plasticity of the pancreatic/islet neural tissues in response to injury.
In summary, we characterised the plasticity of Schwann cells and pericytes in response to islet injury in experimental diabetes. Before this research, Schwann cells and pericytes had been overlooked with regard to their dynamic roles in the lesion during the progression of autoimmune diabetes. This is in part due to the difficulty in observing the islet tissue networks in a space continuum. We used a recently developed 3D imaging technique to resolve the spatial distribution of the tissue networks for qualitative and quantitative analyses of the two cell types to identify their reactive cellular responses in islet injury. Because several classes of molecular inducers and suppressors of glial scar have been recognised through studies on CNS injuries, future studies on manipulation of gliosis in experimental diabetes will further benefit our understanding of the cellular components in the progression of islet lesions in type 1 diabetes.
We thank Ann-Shyn Chiang at the Brain Research Center, National Tsing Hua University for technical support in confocal imaging.
This work was supported in part by grants from the Taiwan National Health Research Institutes and National Science Council to SCT.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
All authors contributed to the experimental conception and design. YCC, CTH, SJP and YYF contributed to data acquisition, analysis and interpretation of data, and revised the manuscript critically for intellectual content. SCT directed the imaging project and contributed to the analysis and interpretation of data and the writing of the paper. All the authors approved the final version of the paper.
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