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. 2017 Dec 6:8:1712.
doi: 10.3389/fimmu.2017.01712. eCollection 2017.

Loss of Sympathetic Nerves in Spleens from Patients with End Stage Sepsis

Donald B Hoover  1   2 , Thomas Christopher Brown  1 , Madeleine K Miller  1 , John B Schweitzer  2   3 , David L Williams  2   4
Affiliations

Loss of Sympathetic Nerves in Spleens from Patients with End Stage Sepsis

Donald B Hoover et al. Front Immunol. .

Abstract

The spleen is an important site for central regulation of immune function by noradrenergic sympathetic nerves, but little is known about this major region of neuroimmune communication in humans. Experimental studies using animal models have established that sympathetic innervation of the spleen is essential for cholinergic anti-inflammatory responses evoked by vagal nerve stimulation, and clinical studies are evaluating this approach for treating inflammatory diseases. Most data on sympathetic nerves in spleen derive from rodent studies, and this work has established that remodeling of sympathetic innervation can occur during inflammation. However, little is known about the effects of sepsis on spleen innervation. Our primary goals were to (i) localize noradrenergic nerves in human spleen by immunohistochemistry for tyrosine hydroxylase (TH), a specific noradrenergic marker, (ii) determine if nerves occur in close apposition to leukocytes, and (iii) determine if splenic sympathetic innervation is altered in patients who died from end stage sepsis. Staining for vesicular acetylcholine transporter (VAChT) was done to screen for cholinergic nerves. Archived paraffin tissue blocks were used. Control samples were obtained from trauma patients or patients who died after hemorrhagic stroke. TH + nerves were associated with arteries and arterioles in all control spleens, occurring in bundles or as nerve fibers. Individual TH + nerve fibers entered the perivascular region where some appeared in close apposition to leukocytes. In marked contrast, spleens from half of the septic patients lacked TH + nerves fibers and the average abundance of TH + nerves for the septic group was only 16% of that for the control group (control: 0.272 ± 0.060% area, n = 6; sepsis: 0.043 ± 0.026% area, n = 8; P < 0.005). All spleens lacked cholinergic innervation. Our results provide definitive evidence for the distribution of noradrenergic nerves in normal human spleen and the first evidence for direct sympathetic innervation of leukocytes in human spleen. We also provide the first evidence for marked loss of noradrenergic nerves in patients who died from sepsis. Such nerve loss could impair neuroimmunomodulation and may not be limited to the spleen.

Keywords: cholinergic; human; innervation; noradrenergic; remodeling; sepsis; spleen.

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Figures

Figure 1
Figure 1
Control human spleens had abundant tyrosine hydroxylase (TH) + sympathetic nerves associated with the vasculature and lacked cholinergic innervation. (A,B) Images showing noradrenergic (TH+) nerve fibers and bundles around splenic artery and branches (A) and absence of cholinergic (VAChT) nerves in an adjacent section (B) from a 74-year-old female. (C) Low magnification image of TH + nerve fibers and bundles around a trabecular artery in a spleen section from a male trauma patient. Arrows indicate TH + nerve fibers around an adjacent large vein. (D,E) TH + fibers associated with central artery and smaller branches in the same 74-year-old female. Nerve fibers are localized to adventitia and adventitia-media border. Arrow in (D) indicates nerve around branch of central artery shown at higher magnification in (E). (F) Sympathetic nerves around a central artery and branch from another trauma patient. Arrows indicate some TH + nerve fibers that entered the periarterial lymphatic sheath. (G) Same patient had TH + nerve fibers (some indicated by arrows) in lymphatic tissue under the capsule. (H,I) TH + fibers around small, branching arteries in sections from first trauma patient (H) and from a 57-year-old female (I). (J) Periarteriolar TH + nerve fibers in spleen section from a 74-year-old female. TH + nerve fibers extend a short distance into surrounding lymphatic tissue. Open arrows indicate some TH + nerve bundles. Scale bars: 250 µm (A–C); 100 µm (D,F–I); 50 µm (E,J).
Figure 2
Figure 2
Sympathetic nerves in perivascular lymphatic tissue of control spleens have close association with leukocytes. (A,B) Hematoxylin and eosin stain of small arteries associated with a splenic nodule (n) in section from a trauma patient (A) and tyrosine hydroxylase (TH) + nerve fibers around same vessels in adjacent section (B). Small boxes indicate regions shown at higher magnification (C,D) to demonstrate close apposition of sympathetic nerves with leukocytes. (E,F) Close apposition of sympathetic nerves with perivascular leukocytes (arrows) in spleen sections from female patients who were 74 and 57 years old, respectively. (G–I) Close apposition of TH + nerve processes with perivascular leukocytes (some indicated by arrows) in spleen section from another trauma patient. Scale bars: 100 µm (A,B); 25 µm (C–I).
Figure 3
Figure 3
Close association of tyrosine hydroxylase (TH) + nerves and leukocytes demonstrated in dual stained sections. (A–F) High magnification images of control spleen sections with TH + nerves stained brown using DAB and nuclei stained blue with hematoxylin to enhance identification of leukocytes. Arrows indicate some TH + nerves in close apposition to leukocytes. Scale bars: 25 µm (A,C–F); 50 µm (B).
Figure 4
Figure 4
Loss of sympathetic nerves in spleens from patients who died with sepsis. (A,B) Hematoxylin and eosin stain (A) and tyrosine hydroxylase (TH) stain (B) of spleen section from 63-year-old male. Note absence of TH + nerve fibers around arteries. (C) Absence of TH + nerves around large artery (*) and smaller artery (a) in spleen section from an 87-year-old female patient. Sections from both of these patients lacked sympathetic nerves. (D–F) Images showing reduced abundance of periarterial TH + nerve fibers in spleen from a 70-year-old female patient who died with sepsis. Large (D) and intermediate (E) diameter arteries show marked reduction of TH + nerve fibers compared to control patients and lacked large TH + nerve bundles. Smaller arteries (F) lacked TH + nerve fibers in this patient. (G) Solitary axon retraction ball in white pulp. (H) Tortuous reactive axonal profile with highly variable axonal diameter. (I) Axonal swelling where apparently intact axon profiles enter and leave zone of interrupted axonal transport. Scale bars: 100 µm (A,B,F); 250 µm (C–E); 25 µm (G–I).
Figure 5
Figure 5
Quantification of tyrosine hydroxylase (TH) nerve fiber staining shows a significant deficit in spleens from patients with end stage sepsis. (A) Semi-quantitative evaluation of TH nerve fiber abundance was performed using a grading scale ranging from 0 to 3 as described in Section “Materials and Methods.” (B) Quantitative evaluation of TH nerve fiber abundance was performed, as described in Section “Materials and Methods,” using ImageJ to measure the % area occupied by TH + nerve fibers. Both methods showed a significant reduction of TH + nerve fibers in spleen samples from septic patients compared to control patients. Data in panel (A) were evaluated using a two-tailed Mann–Whitney test for unpaired samples (n = 6 for control and n = 8 for septic; P = 0.0077). Data in panel (B) were evaluated using a two-tailed t-test for unpaired samples (P = 0.0024). Values are mean ± SD.
Figure 6
Figure 6
VAChT antibody labels cholinergic nerve fibers in human intestine. (A) Low magnification image showing the presence of VAChT + cholinergic nerve fibers in a section of human intestine that was stained along with spleen sections. Asterisks indicate a ganglionated nerve plexus. Boxed region contains a ganglion shown at high magnification in panel (B). Arrows indicates two of many VAChT nerve fibers that innervated intestinal smooth muscle (sm). (B) VAChT + nerve fibers surrounding an enteric ganglion. (C) VAChT + nerve fibers in sm region shown at higher magnification. Scale bars: 100 µm (A); 50 µm (B,C).
Figure 7
Figure 7
Diagram illustrating potential mechanisms contributing to loss of sympathetic nerves in end stage sepsis. Top panel shows normal condition (control) where sympathetic innervation is maintained by nerve growth factor (NGF), which is most likely supplied by T and B cells. The bottom panel depicts changes in sepsis that could contribution to the loss of sympathetic nerves. Loss of lymphocytes in the septic spleen would decrease the supply of NGF, which is needed to maintain sympathetic nerves. Semaphorins, which are nerve repellants, could be generated in the septic spleen and cause retraction of sympathetic nerves through their interaction with the neuropilin-2/plexin A2 receptor complex located on tyrosine hydroxylase (TH) + nerve fibers. Toxic free radicals and inflammatory cytokines, generated locally during sepsis, might contribute to degeneration of TH + nerve fibers.
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