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Abstract
Henoch–Schönlein purpura (HSP) is a typical cutaneous immune skin disease, usually diagnosed by invasive biopsy. In this study, we develop a noninvasive optical method by combining in vivo optical clearing, confocal microscopy and immune-staining together to present the real-time in vivo dynamics of blood vessels, IgA molecules, and T cells in a HSP rat model. The small vessels in the skin are found with acute damage and then hyperplasia, which enhances deposition of IgA complexes in blood vessels. The migrating T cells in blood vessels in HSP regions can be detected by setting fast line scanning in this method. Our method provides in vivo vascular, cellular, and molecular dynamics during HSP development and is thus of great potential in research and diagnosis of HSP and other skin diseases.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Skin is the first and most important barrier to physically defense against injury, insult, and infections of environmental microbes. The blood vessels and cells in dermis further provide functional immunebarrier. Henoch–Schönlein purpura (HSP), also named as small-vessel vasculitis or IgA vasculitis, is a typical cutaneous immune disease, usually induced by infections or drugs [1–3]. The vasculitis, commonly with manifestation of cutaneous purpura, can simultaneously develop in heart, intestinal tract, and particularly kidney that may finally develop to renal failure [4,5].
The essential immunol change in HSP is deposition of immune-complexes, mainly IgA, onto the endothelium of small vessels in skin or glomerulus of kidney. It is implicated in the recruitment of immune cells and their infiltration and extravasation through small vessels, by which the permeability of the vascular wall is influenced to develop vasculitis [6,7]. The corresponding organs are affected. In current stage, the clinical diagnosis and medical research of HSP are generally based on invasive biopsy and pathological images of the tissue sections [8–10]. However, the real-time and continuous molecular dynamics during HSP development are unclear. The in vitro biopsy method might miss some real-time information in the immune processes in blood vessels since the immune molecular level in blood might vary significantly and rapidly during HSP development [11,12]. The IgA deposition dynamics in blood vessels and their role in mediating vasculitis generation remain obscure.
Skin tissue, the surface of body, is naturally suitable with optical microscopy. It has been demonstrated that confocal and multiphoton microscopy can detect the autofluorescence from endogenous chromophores in skin [13–16]. The microscopy depth can be improved by in vivo optical clearing agents [17,18]. Series of works report the successful diagnostics of skin disease by in vivo optical microscopy [19–21]. Basal cell carcinoma and malignant melanoma are found able to be distinguished effectively by multiphoton microscopy of the autofluorescence from melanin or collagen in skin [22–24]. Recently, the in vivo microscopy on skin aging is also reported with the imaging depth at tissue level [25–28]. Therefore, optical microscopy is shown as a powerful technology for in vivo imaging of skin tissue. But all those studies are based on the autofluorescence from endogenous fluorophores (like second harmonic generation from collagen) in skin that mainly reveals the structural information of skin but no information of immune molecules.
In this study, we report an optical scheme of in vivo immunofluorescent microscopy and present the real-time in vivo dynamics of IgA molecules and T cells in blood vessels in a HSP rat model. Our results provide an optical noninvasive biosensor scheme to the research of pathogenesis, diagnosis, and therapy of HSP.
2. Materials and methods
2.1 Establishment of the HSP rat model
The HSP rat model was established by referring protocols in previous studies [29]. It took 36 days in total. Briefly, at Phase 1 (the first 3 weeks), ginger, long pepper, pepper decoction (Beijing Tong Ren Tang Chinese Medicine Co., Ltd) were mixed at a 1:1:1 ratio in an aqueous solution (15 g/100 mL), incubated in a water bath at 60$\mathrm{\circ{C}}$ for 30 minutes, and filtered. The intragastric administration of this solution to the model rats at 0.2 mL-0.4 mL every day for the first 3 weeks (Day 1-21). At Phase 2 (the last 2 weeks), those rats were intraperitoneally injected with 0.1-0.4 mL ovalbumin (OVA) (F5503; Sigma) emulsified solution (10 mg/kg, mixture of OVA (20 mg/mL) and Freund’s complete adjuvant (F5881; Sigma) at 1:1) once per week at the beginning of each week (Day 22, 29, and 36). At the last day (Day 36), those rats were additionally stimulated by OVA physiological saline liquor (10 mg/mL) through tail vein injection (0.25 mL) and intradermal injection (1 mL) simultaneously. The bleeding spots exhibited at the intradermal injection points after around 0-12 hours. The rats in control received the same injections of equal amounts of physiological saline. All efforts were made to reduce animal suffering. All animal experiments were performed in accordance with guidelines evaluated and approved by the Ethical Committee of Animal Experiments, School of Biomedical Engineering, Shanghai Jiao Tong University, China. The study was approved by the Ethic Committee of the School of Biomedical Engineering at Shanghai Jiao Tong University (202101332).
2.2 In vivo fluorescent microscopy
The blood vessels and IgA molecules in HSP rats were fluorescently indicated by intravenous injection of FITC (Goat Anti-Rat IgA alpha chain, ab97184, Abcam) and IgA-PE (Anti-Mouse IgA PE, 12-5994-81, eBioscience) respectively one hour before in vivo fluorescent microscopy. At the same time, the hair at the targeted regions (the intradermal injection locations) was shaved. After 30 minutes, optical clearing was performed at that region by smearing the skin with 30% glycerol at 38 $\mathrm{\circ{C}}$. In this study, glycerol, a nonirritating reagent, was adopted for HSP rats considering it could protect the HSP skin with bleeding spots and inflammation from further damage [30]. Some other optical clearing reagents, like alcohol, could greatly irritate skin tissue, especially the HSP skin with significant vasculitis.
After another 30 minutes, in vivo confocal microscopy was performed for imaging of the blood vessels and IgA molecules under the skin. For the imaging of FITC, the excitation laser wavelength was 488 nm and the power was around 3-5 mW. The fluorescence was collected by a 20${\times} $ objective (N.A. = 0.9, water immersed) and filtered by a bandpass filter at 490-550 nm. For the imaging of IgA-PE, the excitation laser wavelength was 543 nm and the power was 2 mW. The fluorescence was detected at 560-610 nm. The two channels were split by a dichroic mirror at 560 nm. Usually in the in vivo microscopy of blood vessels, the dwell time of each pixel was set as 2 μs. The images were defined as 512${\times} $ 512 pixels. The imaging speed was 1.1 s/frame. For the real-time microscopy of flowing cells in blood vessels, the image was defined as 128 ${\times} $ 100 pixels with dwell time of 2 μs/pixel. The imaging speed was 24 frames/s.
2.3 Image processing
The length and width of blood vessels were acquired by processing the fluorescent images by ImageJ. Briefly, the morphology of vessels was extracted from the background by setting a threshold of the FITC fluorescence intensity. The autofluorescence from hairs which presented both very bright green and red fluorescence and distinct shape from vessels was removed manually. The length and width of vessels could then be acquired.
To calculate the curvature of vessels, after acquiring the vascular morphology, the blood vessels in each image were calculated by using the plugin “Kappa” of ImageJ. After fitting the curve, the curvature at each point in the vessels could be given. In this study, we only selected the top 50 curvature values in each image to represent the curvature of the blood vessels in each field of views.
3. Results
3.1 Immunofluorescence of IgA by biopsy from a HSP rat model
We established HSP rat model by sensitization with OVA that took 36 days according to the protocol described as the Materials and Methods section. The abdominal skin treated with the medicine exhibited with tiny bleeding spots and purpura, indicating the emergence of HSP, as shown in Fig. 1(a). To confirm it, we extracted the skin biopsy from the drug-treated skin area and performed the IgA immunofluorescence assay. The positive IgA fluorescence signals could be found in the blood vessels in the sections (Fig. 1(b)), suggesting the vasculitis had already existed in the skin tissue.
Fig. 1. The establishment of the rat HSP model. (a) The bleeding spot on the rat skin after the establishment of HSP model. Bar: 1 cm. (b) The immunofluorescence of IgA of the skin section by biopsy of the rat. Arrows: the IgA signal. Bar: 20 μm.(c) The experimental design. Immediately after the establishment of HSP model (two phases for 36 days), the rats were observed by in vivo microscopy. Insert: the procedure of in vivo microscopy. It took 1 hour for preparation and fluorescence labeling.
The HSP skin areas were then observed in vivo immediately after the establishment of HSP model and continuously as designed in Fig. 1(c). The rat hair on the vasculitis region was shaved. The IgA antibody and fluorophores to label blood vessels were injected to the rats by intravenous injection 1 hour before microscopy. The skin was then smeared with 30% glycerol at 38 $\mathrm{\circ{C}}$ for optical clearing 30 minutes before microscopy. The blood vessels in dermis were observed at 0, 12, 24, and 44 hours after the establishment of HSP model.
3.2 In vivo fluorescent microscopy of IgA and blood vessels during HSP
We performed confocal microscopy aided with optical clearing for the in vivo microscopy of blood vessels and IgA molecules. The blood vessels and IgA molecules were excited serially to prevent crosstalk between the green and red fluorescence channels. The two-photon microscopy was not adopted to prevent the crosstalk between the green (490-550 nm) and red (560-610 nm) fluorescence channels since the femtosecond laser could excite the green and red fluorescence simultaneously. We examined the fluorescent signals from healthy rats in control by the in vivo microscopy scheme. As shown in Fig. 2(a), the autofluorescence from the skin, generally from the keratin, flavin, and melanin in the deeper layer of epidermis (the basal layer or stratum spinosm, at around 40-70 μm deep from the surface), could be detected. It should be noted that the hairs, excited by the 488 nm laser, emitted very bright autofluorescence at both green and red bands. Fortunately, the hairs could be distinguished easily according to the morphology. No signals from blood vessels or IgA could be found.
Fig. 2. The in vivo fluorescent microscopy of the healthy rats as control. (a) The autofluorescence from the basal layer or stratum spinosm at the bottom of epidermis, at around 60 μm deep from the surface. Arrows: autofluorescence from the hairs. (b) The in vivo microscopy at 150 μm deep. Dashed box: blood vessels indicated by FITC. Arrows in the Merge: blood vessels without any red fluorescence. Bar: 100 μm.
At the depth of 150 μm, blood vessels at the upper layer of dermis indicated by FITC in the green channel could be observed (Fig. 2(b)). The network of blood vessels is in layers in dermis with large interval between them. The first vascular layer in dermis at 150-300 μm deep could be observed in this way. The blood vessels were not too many in those healthy rats. No IgA signals were found in those vessels.
The HSP rats were then observed continuously by the in vivo microscopy method after the establishment of HSP model. At 0 h, with the aid of optical clearing, a mess of squiggly blood vessels were found at 125 μm deep with very high density and totally different morphology as in Fig. 3(a) and Visualization 1. Without in vivo optical clearing, the vascular fluorescence could hardly be detected. Notably, the IgA immunofluorescence signals could be found in the blood vessels, suggesting the deposition of IgA there. Then, those small vessels were hyperplastic and developed towards the surface at 12 hours after the HSP establishment. The hyperplastic vessels could be observed clearly in the microscopy region at 85 μm deep (Fig. 3(b)), where no blood vessels could be found in control at such a shallow imaging plane. The deposition of IgA could be found in most of the blood vessels, exhibiting bright fluorescence signals along the vessels. At 24 h, the elongated vessels tended relatively straight and could still be observed at 80 μm deep (Fig. 3(c)). The permeability of blood vessels was influenced significantly such that the FITC molecules in the blood leaked out and presented hollow fluorescence (the insert in Fig. 3(c)). The IgA molecules still remained in the vessels, but the fluorescence showed fragmented distributions probably due to the uneven deposition of IgA that further induced permeability damage of the vessel walls and the leakage of IgA or fluorophores. Marked leakage of these vessels caused the visible purpura of HSP skin lesions. At 44 h, the vessels could still be found at 80 μm deep (Fig. 3(d)). The FITC fluorescence was uniform in the vessel structure, suggesting the repair and recovery of the vessel wall. The reason should be the IgA depletion in the blood vessels as shown in Fig. 3(d). This result is consistent with the natural period of HSP development.
Fig. 3. In vivo microscopy of rat HSP model. The fluorescent images of blood vessels (green, indicated by FITC) and IgA (red, indicated by IgA-PE) at 0 h (a), 12 h (b), 24 h (c), and 44 h (d). Arrows in (a-c), the IgA deposition in blood vessels in red channel. Red dashed box in (c): the blood vessels magnified in the insert. Arrows in (d): blood vessels without any IgA deposition. Bar: 100 μm.
We quantified the morphology of blood vessels and IgA deposition dynamics during the HSP development. The average total length of blood vessels in a random microscopy region (500 ${\times} $ 500 μm2), i.e., the density of vessels, was significantly higher in the HSP rats than in the control animals. The total length of blood vessels in the field of view reached the maximum at 12 h (Fig. 4(a)). At 44 h, the length decreased, but still significantly longer than it of control. The largest diameter of the vessels was found at 0 h and decreased at 24 h (Fig. 4(b)). We calculated the curvature radius of the blood vessels to quantify the vascular winding level. The curvature radius was quite small at 0 and 12 h, but recovered closely to normal at 24 h (Fig. 4(c)). Probably the vasculitis initiated since the beginning (0 h) and the vascular hyperplasia remained serious in the first 12 hours.
Fig. 4. The quantified characters of blood vessels. (a) The average total length, (b) the average diameter, and (c) the average curvature radius of blood vessels in the microscopy region (500 ${\times} $ 500 μm2) at different time slots. n = 6 rats, each point was calculated from 5 randomly-selected microscopy regions. N.S., no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by one-tailed students’ t-test.
The IgA fluorescence intensity was significantly higher than it of control since 0 h and recovered back to normal at 44 h as shown in Fig. 5(a), similar to the result of vascular length. The overlap ratio of IgA deposition and blood vessels changed in the same way (Fig. 5(b)). The IgA intensity and overlap ratio both reached the maximum at 12 h. Taken together, those results suggest the vascular lesion further enhances the IgA deposition, and IgA in turn induced more hyperplasia of blood vessels.
Fig. 5. The quantified IgA immunofluorescence. (a) The average fluorescence intensity of IgA in blood vessels. (b) The overlap ratio of IgA fluorescence and blood vessels. n = 6 rats, each point was calculated from 5 randomly-selected microscopy regions. N.S., no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by one-tailed students’ t-test.
3.3 In vivo detection of flowing T cells in blood vessels during HSP
Immune cells have been previously found in the skin tissue in HSP patients in clinical diagnostics. But the migration mechanism remains unknown. We demonstrate here that this in vivo microscopy method could reveal the dynamics of T cells in the blood flow in HSP rats model. We optimized the imaging speed by selecting a small region of interest (100 μm ${\times} $ 80 μm) that was mapped to 128 ${\times} $ 100 pixels, as shown in Fig. 6(a). We injected anti-CD4 immuno-fluorophores (ab196147, Abcam) into rats to label the CD4+ T cells. After 1 hour, the blood vessels in the HSP region were observed by the in vivo microscopy system. Under the excitation by the laser at 650 nm, the 2-dimensional fluorescent images of the flowing CD4+ T cells could be detected. However, the dwell time could not be further shorten for integrating the fluorescence photons. The imaging speed is not high enough to acquire all passing T cells. To this challenge, we further designed a fast single-line laser scanning across the blood vessel along time to cover all passing CD4+ T cells as shown in Fig. 6(b). It took only around 3 ms for a single-time line scanning by setting a 20-μm traverse line across the blood vessel that was mapped to 128 pixels for continuous time-lapse line scanning. This imaging method could thus provide a real-time (333 lines/s) monitoring of the flowing T cells in blood vessels. The spatiotemporal image of the detected T cells, as shown in Fig. 6(b), could provide the precise in vivo dynamics of T cells in the HSP rats. In this way, we measured the kinetics of T cells in the HSP region. As in Fig. 6(c), the CD4+ T cells were quite rare in control. After HSP establishment, the T cells reached the peak at 12 h and recovered to normal at 44 h. Our results implied the T cells in HSP rats probably migrated by blood vessels to recruit IgA molecules and induce the damage of vascular permeability at 12 h to 24 h.
Fig. 6. The kinetics of CD4+ T cells in blood vessels in HSP region. (a) The in vivo fluorescent images of CD4+ T cells in the blood flow in dermis in HSP region. Right panel: the real-time dynamics of a CD4+ T cell (arrows) in blood vessels (dashed lines) at different times. (b) The fast line scanning method. Right panel: the x-t image of T cells. Arrows: the detected T cells. Dashed line: the boundary of the blood vessel. (c) The kinetics of T cells along time during the HSP development. * p < 0.05, ** p < 0.01, by one-tailed students’ t-test.
4. Discussion
By this in vivo microscopy method, the real-time dynamics of blood vessels and IgA deposition during HSP development could be observed in vivo noninvasively. The vasculitis was found involved with dramatic vascular morphological transformation and IgA deposition there. The small vessels at the first layer in dermis presented significant vascular hyperplasia immediately after the establishment of HSP rat model. The vascular diameter and curvature radius, which indicated the vascular lesion, did not change synchronously with hyperplasia. By comparing the dynamics of blood vessels and IgA (Fig. 4 and 5), it could be found the IgA deposition and hyperplasia developed synchronously and reached the maximal level at 12 h, with a delay of around 12 hours to the vascular lesion at 0 h. These results imply that the vascular lesion enhances IgA deposition. The high overlap ratio of IgA fluorescence and blood vessels in Fig. 5(b) further presents the IgA molecules remained to deposit on the vessel wall at 24 h, although the fluorescence intensity decreased at that time (Fig. 5(a)). The decrease of IgA fluorescence at 24 h could be explained by the leakage of blood vessels, as suggested in Fig. 3(c). Therefore, the IgA deposition is highly relative to the hyperplasia. The T cells could migrate to the HSP region from 12 h to 24 h, suggested by the kinetic data of T cells in the blood flow and leakage of the blood vessels.
To prevent crosstalk between the green and red channels, we did not use multiphoton microscopy, which could simultaneously excite the fluorescence of FITC and IgA-PE by the femtosecond laser, although the microscopy depth could be improved. In this study, we adopted confocal microscopy to excite FITC by a 488-nm laser and IgA-PE by a 543-nm laser. Before microscopy, we tested the crosstalk of the fluorescence by injecting only one single fluorophore into rats through tail vein respectively. If injecting only FITC and exciting it by the 488-nm laser, no red fluorescence could be detected in the red channel. Meanwhile, after i.v. injection of IgA-PE, no green fluorescence was detected in the green channel when excited by the 543-nm laser. In experiments, we also performed microscopy of FITC and IgA-PE separately.
During HSP, immune cells concentrate in the blood flow in the HSP region and invade the vascular wall. Our method can detect these immune cells in vivo. The immune cells in blood flow require very fast imaging speed due to their fast velocity. The integral duration for fluorescence collection is quite short. This difficulty can be overwhelmed by high-sensitive photodetectors. Another challenge is the in vivo immunolabeling. Although the intravenous injection of the antibody of T cells works in blood vessels, the antibody molecules can hardly diffuse from blood vessels out to label the immune cells in the interstitial tissue.
In this study, we present a scheme of in vivo microscopy to observe the blood vessels, IgA molecules, and T cells inside noninvasively. This scheme includes in vivo fluorescence labeling, optical clearing, and fluorescent microscopy. It can reveal the continuous dynamics of blood vessels, IgA molecules, and T cells in dermis of rat HSP model. Our results suggest the vascular transformation enhances IgA deposition, which is further involved in hyperplasia. The development of IgA vasculitis is highly related to the morphological transformation of blood vessels. The T cells could migrate to the HSP region by blood flow. Our method thus provides a powerful tool for in vivo research of skin disease and potentially brings deeper insights in the molecular dynamics in immune processes of skin.
Funding
National Natural Science Foundation of China (61975118, 62022056, 81730085); Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JYZZ129).
Acknowledgments
We thank Yutong Wang for the help on animal preparation and microscopy.
Disclosures
The authors declare no conflict of interest.
Data Availability
Data underlying the results presented in this study are available in this paper. The data now shown may be obtained from the authors upon reasonable request.
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