The unsectioned mouse or human prostate is processed for the whole-tissue immunolabeling based on the reported iDISCO+ protocol (Renier et al., 2016). Additional optimizations are included as we recently published (Cao and Yang, 2020). The fresh tissues were collected and fixed in 1% paraformaldehyde/10% sucrose/phosphate-buffered saline at room temperature for 60 min and further fixed in 1% paraformaldehyde/phosphate-buffered saline for 240 min. After the fixation, the tissues were washed with phosphate-buffered saline at room temperature for 60 min three times. The tissues were processed through a methanol gradient (MeOH diluted in ddH₂O) at room temperature: 20% for 120 min, 40% for 120 min, 60% for 120 min, 80% for 120 min, and 100% for 120 min. The tissues were decolorized for 2 days at 4°C with 3% H₂O₂ diluted in 100% MeOH. The tissues were then processed through a reverse MeOH gradient at room temperature: 100% for 120 min, 80% for 120 min, 60% for 120 min, 40% for 120 min, and 20% for 120 min. The tissues were permeabilized with 0.1% Deoxycholate/0.2% TritonX-100/10% DMSO/10 mM EDTA/phosphate-buffered saline at room temperature for 24 h.

The tissues were incubated with 0.2% TritonX-100/10% DMSO/phosphate-buffered saline/5% normal donkey serum at room temperature for 24 h. The tissues were then immunolabeled with the primary antibodies (final concentration of 2 μg/ml) in 0.1% Tween-20/5% normal donkey serum/phosphate-buffered saline at room temperature for 3 days. The primary antibodies for the whole-tissue immunolabeling included rabbit anti-PGP9.5 (Proteintech, 14730-1-AP), rat anti-PECAM1 (BD Biosciences, 553370), goat anti-VEGFR3 (R&D Systems, AF743), rabbit anti-TH (Millipore, AB152), and goat anti-VAChT (Millipore, ABN100). The tissues were washed with 0.1% Tween-20/10 μg/ml heparin/phosphate-buffered saline at room temperature for 6 h four times. The tissues were then immunolabeled with the Alexa Fluor dye-conjugated secondary antibodies (Thermo Fisher Scientific, final concentration of 4 μg/ml) in 0.1% Tween-20/5% normal donkey serum/phosphate-buffered saline at room temperature for 3 days. The tissues were washed with 0.1% Tween-20/10 μg/ml heparin/phosphate-buffered saline at 37°C for 8 h six times.

Before the optical-clearing steps, the immunolabeled prostate tissues were made into 0.8% agarose blocks. The tissue blocks were processed through a MeOH gradient at room temperature: 20% for 60 min three times, 40% for 120 min, 60% for 120 min, 80% for 120 min, 100% for 120 min twice, and 100% for 12 h. The tissue blocks were delipidated at room temperature with 50% dichloromethane/50% MeOH for 60 min three times, followed by 100% dichloromethane for 60 min three times. The tissue blocks were optically cleared at room temperature with 100% dibenzyl ether for 12 h twice.

After the immunolabeling and optical clearing, the prostate tissues were scanned by the LaVision Biotec Ultramicroscope II with a 2x/NA0.5 objective. For imaging at high magnification (1.26x), each tissue was scanned with the 4-μm z-step by three lightsheets illuminating from the left side. For imaging at low magnification (12.6x), each tissue was scanned with the 1-μm z-step by a single lightsheet illuminating from the left side.

The image stacks obtained from the lightsheet imaging were 3D reconstructed by Imaris.¹ For the display purpose in the figures and movies, a gamma correction (1.3∼1.6) was applied. The representative 3D images in the figures were produced by orthogonal projection. The movies were filmed with 30 frames per second.

Human prostate tissues were collected in compliance with the protocol approved by the Institutional Ethics Committee of Peking University People’s Hospital, and informed consent was signed by each patient. The normal prostate tissues were sampled from three male patients during the transurethral resection of prostate cancer.

The mouse experiments were conducted in compliance with the protocol approved by the Institutional Animal Care and Use Committee of Peking University.

C57BL/6 mice were purchased from Charles River International. Th-Cre (008601) and TrkA^fl/fl (022362) mice were obtained from Jackson Laboratory and in-house bred to generate Th-Cre; TrkA^fl/fl and control Th-Cre; TrkA^+/+ littermates. Mice were housed on the 12 h/12 h light cycle, and a chow diet and water were provided ad libitum. Male mice of 8 weeks old were utilized in the experiments unless otherwise specified.

For the castration procedure, mice were anesthetized with 3% isoflurane. A midline incision was made along the skin region of the scrotum. The testes, vas deferens, and the attached connective tissues on both sides were pulled out. After ligating the blood vessels supplying each testis with a 5/0 suture thread, the testes on both sides were removed. The skin incision was then closed by wound clips. The sham surgery was performed with all the steps except the ligation and removal of testes.

For the 6-OHDA treatment, each mouse was intraperitoneally injected with 2 mg 6-OHDA (Sigma, prepared in 200 μl 0.1% ascorbic acid/phosphate-buffered saline). For the antibiotic treatment, each mouse was administered daily with a combination of ampicillin, neomycin, vancomycin, and metronidazole (together prepared in 200 μl sterile saline) at 10 mg/kg of body weight via intraperitoneal injection for 5 days.

For the treatment of the α1-adrenergic receptor antagonist, each mouse was administered daily with prazosin (prepared in 200 μl sterile saline) at 5 mg/kg of body weight via intraperitoneal injection for 14 days. For the treatment of the α1-adrenergic or β2-adrenergic receptor agonists, each mouse was administered daily with phenylephrine, clenbuterol, or formoterol (prepared in 200 μl sterile saline) at 5 mg/kg of body weight via intraperitoneal injection for 5 days.

Mouse anterior and ventral prostate lobes were utilized for analyses due to their relatively larger tissue size compared to the lateral or dorsal lobes. For the qPCR analysis of mRNA levels, both sides of the anterior or ventral prostate of each mouse were harvested and pooled. Total RNAs were purified using the RNeasy Mini Kit (Qiagen). mRNA levels were analyzed by the SYBR Green Real-Time PCR Kit (Thermo Fisher Scientific), with Cyclophilin mRNA levels being used as the internal control.

For the fluorescence-activated cell sorting (FACS) analysis, both sides of the anterior prostate of each mouse were harvested and pooled. The tissues were digested in Hanks’ balanced salt solution (HBSS) containing 3% heat-inactivated fetal bovine serum/20 μg/ml DNase I/10 μg/ml Liberase (Roche) at 37°C for 20 min and then mashed by a 40-μm cell-strainer. The cells were centrifuged at 1,000 g for 10 min and re-suspended in HBSS containing 3% heat-inactivated fetal bovine serum. The cells were stained by FACS antibodies and analyzed on the BD LSRFortessa. In addition, the FACS-stained CD45⁺CD11b⁺F4/80⁺ macrophages were collected on the BD FACSAria for the qPCR analysis of mRNA levels.

For the enzyme-linked immunosorbent assay (ELISA), both sides of the anterior prostate of five saline-treated or 6-OHDA-treated mice were harvested and pooled. The tissues were thoroughly homogenized in 4 ml phosphate-buffered saline containing Protease Inhibitor Cocktail (Roche). A final concentration of 0.1% Tween-20 was then added to the homogenates and incubated at room temperature for 15 min. The homogenates were centrifuged at 20,000 g for 10 min to clear the tissue debris. The supernatants were filtered through a 0.22-μm PES filter and analyzed by Proteome Profiler Mouse Cytokine Array (R&D Systems).

Tissue volumes were measured in the reconstructed 3D images by Imaris. For the quantification of neural innervations, four 200 μm × 200 μm × 200 μm volumes were randomly chosen in the reconstructed 3D images, and axons in each volume were manually measured.

All the experiments were repeated at least twice, with consistent results obtained. GraphPad Prism² performed Student’s t-test or ANOVA test.

We aimed at a comprehensive 3D assessment of neural anatomy in the prostate. We pursued the protocol based on the iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs plus) method (see Section “Materials and Methods”). Importantly, this imaging procedure supports the whole-tissue immunolabeling and optical clearing of prostate tissues. The entire prostate from the adult mouse appeared completely transparent after the processing steps (Figure 1A), thus enabling the lightsheet 3D imaging.

We first subjected the mouse prostate to the whole-tissue immunolabeling of PGP9.5 (protein gene product 9.5), a specific pan-neural marker. The neural architecture in the mouse prostate was revealed for the first time in the field (Figure 1B and Supplementary Video 1). The high-magnification lightsheet optical sections and 3D images showed the neural inputs to the regions such as the anterior and ventral prostate lobes at single-fiber resolution (Figure 1C). Notably, the density of PGP9.5-positive total axons in the ventral prostate (1228 ± 101 mm/mm³) was approximately 50% higher than that in the anterior prostate (780 ± 41 mm/mm³). As an aside, this 3D imaging technique was also able to visualize different cellular structures in addition to neural innervations. For instance, the whole-tissue immunolabeling of PECAM1 (platelet endothelial cell adhesion molecule 1), a marker for vascular endothelial cells, exhibited the network of blood vessels in the prostate (Figures 1D,E and Supplementary Video 2). Similarly, the whole-tissue immunolabeling of VEGFR3 (vascular endothelial growth factor receptor 3), a marker for lymphatic endothelial cells, demonstrated the 3D distribution of lymphatic vessels in the prostate (Figures 1F,G).

We further analyzed the 3D neural distribution of the human prostate. The unsectioned prostate tissue from the adult human male was rendered nearly transparent by the advanced imaging procedure (Figure 2A). The whole-tissue PGP9.5-immunolabeling revealed dense neural innervations throughout the human prostate (Figure 2B). Interestingly, neural innervations beneath the prostate capsule projected parallel while those within the gland region were organized around the acini (Figure 2C). Also, the whole-tissue immunolabeling of TH (tyrosine hydroxylase), a marker for sympathetic innervations, showed the abundant presence of sympathetic distributions within the human prostate (Figure 2D). TH-positive sympathetic axons also exhibited the parallel distribution beneath the capsule but the concentric pattern in the gland region (Figure 2E).

We examined the status of neural innervations in the mouse prostate after castration, a procedure known to induce significant tissue regression. The tissue volumes of the anterior and ventral prostate of the mice receiving sham surgery were 24.67 ± 1.72 mm³ and 6.08 ± 0.33 mm³, respectively. 4 weeks after castration, the tissue volumes of the anterior (2.53 ± 0.24 mm³) and ventral prostate (0.64 ± 0.08 mm³) decreased by approximately 90% (Figure 3G). However, the whole-tissue immunolabeling of PGP9.5 suggested a dramatic increase in axonal density within the prostate (Figures 3A,B). In particular, PGP9.5-positive total axons in the anterior prostate were 780 ± 41 mm/mm³ in the sham mice but became 7671 ± 619 mm/mm³ in the castrated mice. Also, PGP9.5-positive total axons in the ventral prostate increased from 1228 ± 101 mm/mm³ in the sham mice to 11703 ± 1104 mm/mm³ in the castrated mice (Figure 3H). As a result, the total length of PGP9.5-positive axons calculated in the anterior and ventral prostate appeared unaffected between the sham and castration conditions (Figure 3I). Moreover, the whole-tissue TH-immunolabeling showed a similar increased density of sympathetic innervations in the prostate after castration (Figures 3C,D,H) and the stability of sympathetic innervations after castration (Figure 3I). At the same time, the whole-tissue immunolabeling of VAChT (anti-vesicular acetylcholine transporter), a specific parasympathetic marker, also demonstrated the increase of parasympathetic inputs to the prostate during castration-induced tissue regression (Figures 3E,F,H). Accordingly, the total length of VAChT-positive parasympathetic innervations was comparable between the sham and castration conditions (Figure 3I).

Aided by the advanced imaging technique, we focused on the sympathetic innervations in the prostate. We first exploited the pharmacologic sympathetic ablation by the intraperitoneal treatment of 6-OHDA (6-hydroxydopamine). The whole-tissue TH-immunolabeling and 3D imaging exhibited the effective removal of sympathetic innervations in the prostate 2 weeks after the 6-OHDA treatment (Figures 4A,B). TH-positive sympathetic inputs to the anterior prostate were 547 ± 56 mm/mm³ in the saline-treated control but became 19 ± 10 mm/mm³ in the 6-OHDA-treated condition. Sympathetic innervations in the ventral prostate also decreased from 737 ± 64 mm/mm³ in the control condition to 33 ± 17 mm/mm³ after the 6-OHDA treatment (Figure 4D). Notably, such pharmacologic sympathetic ablation did not alter the general anatomy or tissue volume of the prostate (Figure 4C). As expected, the whole-tissue VAChT-immunolabeling showed that parasympathetic innervations were unaffected in the 6-OHDA-treated prostate (Figure 4D).

Of importance, we observed that the pharmacologic destruction of sympathetic innervations led to spontaneous inflammation in the prostate. Significant up-regulation of pro-inflammatory cytokines TNFa and IL-6 occurred in the anterior and ventral prostate (Figure 4E). At the same time, expression levels of pro-inflammatory chemokines CCL2 and CCL3 increased in the 6-OHDA-treated prostate (Figure 4E). These pro-inflammatory factors have been indicated in the disease process of chronic non-bacterial prostatitis/CPPS (John et al., 2001; Orhan et al., 2001; Thumbikat et al., 2010). Such increased levels of pro-inflammatory cytokines and chemokines in the 6-OHDA-treated prostate were confirmed by the ELISA on the mouse cytokine array (Figure 4F). We then tested whether this prostate inflammation caused by sympathetic ablation would respond to antibiotics. The combinatorial treatment of four antibiotics (i.e., ampicillin, neomycin, vancomycin, and metronidazole) did not mitigate the increased pro-inflammatory cytokines and chemokines in the 6-OHDA-treated prostate (Figure 4G), ruling out the involvement of any bacterial infection due to the intraperitoneal injection procedure. Such irresponsiveness to antibiotics recapitulated another key feature of chronic non-bacterial prostatitis/CPPS.

We went on to validate the observation that loss of sympathetic inputs would produce sterile inflammation in the prostate. TrkA (tyrosine kinase receptor A) is one of the central neurotrophin receptors for establishing sympathetic innervations (Kuruvilla et al., 2004). We found that the specific deletion of TrkA in sympathetic neurons in Th-Cre; TrkA^fl/fl mice resulted in the significant loss of TH-positive sympathetic axons in the prostate (Figures 5A,D). The lateral and dorsal prostate lobes seemed less sensitive to such sympathetic ablation in Th-Cre; TrkA^fl/fl mice, though the underlying reason is not immediately clear. On the other side, VAChT-positive parasympathetic axons were not affected in Th-Cre; TrkA^fl/fl mice (Figures 5B,D). We noted a prior study reporting that prostate development might be affected by the loss of autonomic neural signals (McVary et al., 1994). However, the general anatomy and tissue volume of the Th-Cre; TrkA^fl/fl prostate remained normal compared to that in the Th-Cre; TrkA^+/+ control prostate (Figure 5C). Importantly, this genetic loss of sympathetic innervations caused a spontaneous inflammation in the Th-Cre; TrkA^fl/fl prostate, as characterized by the up-regulation of pro-inflammatory cytokines and chemokines (Figure 5E), similar to that triggered by the pharmacologic sympathetic ablation.

We then investigated adrenergic signaling downstream of sympathetic inputs in the prostate. Previous studies have mainly focused on the functions of α1-adrenergic receptors in the prostate. In particular, the α1-adrenergic receptor antagonist prazosin has been commonly utilized for treating benign prostatic hyperplasia (Forray et al., 1994; Roehrborn and Schwinn, 2004; Schwinn and Roehrborn, 2008). We confirmed the abundant expression of the α1-adrenergic receptor Adra1a in the mouse prostate (Figure 6A). However, unlike that observed above with loss of sympathetic innervations, blockage of the α1-adrenergic receptor signal with prazosin failed to produce the inflammatory condition in the prostate (Figure 6B). In addition, activation of the α1-adrenergic receptor by the specific agonist phenylephrine did not affect the prostate inflammation caused by the sympathetic ablation (Figure 6C). Therefore, we looked into the potential involvement of other adrenergic receptors. The expression profiling showed that the β2-adrenergic receptor Adrb2 was also highly expressed in the prostate (Figure 6A). More importantly, we demonstrated that treatment of the β2-adrenergic receptor agonists clenbuterol or formoterol was sufficient to suppress the pro-inflammatory cytokines and chemokines in the 6-OHDA-treated prostate (Figure 6C), thus identifying an essential role of β2-adrenergic receptor signaling in this disease context. Finally, we explored the immune cells potentially mediating the sympathetic signal in the prostate. The FACS analysis showed an increased accumulation of CD11b⁺F4/80⁺ macrophages in the 6-OHDA-treated prostate compared to that in the saline-treated control (Figure 6D). Further, these CD11b⁺F4/80⁺ macrophages exhibited a high expression of Adrb2 (Figure 6E), implicating their direct participation in prostate inflammation.