Expression of Somatostatin Receptors 1 and 2 in Human Choroid Plexus and Arachnoid Granulations Implications for Idiopathic Intracranial Hypertension

LABORATORY SCIENCES Expression of Somatostatin Receptors 1 and 2 in Human Choroid Plexus and Arachnoid Granulations Implications for Idiopathic Intracranial Hypertension

Steven E. Katz, MD; Dino D. Klisovic, MD; M. Sue O’Dorisio, MD; Richard Lynch, MD; Martin Lubow, MD

Objective: To investigate the localization of somato- Conclusions: Analogous to their demonstration and to statin receptor types 1 and 2 in human choroid plexus their function in kidney and ocular tissues, these recep- (CP) and arachnoid granulations (AGs) by immunohis- tors may be involved in the processes of cerebrospinal tochemistry. fluid production and absorption, and may play a role in the increased intracranial pressure of idiopathic intra- Methods: A prospective study was performed in an in- cranial hypertension. stitutional setting. Immunohistochemistry was performed on 15 samples of CP and 12 samples of AGs from Clinical Relevance: Somatostatin analogues have been 15 patients who died with no signs or symptoms of in- used to treat idiopathic intracranial hypertension, a dis- tracranial disease (age range, 52-81 years). The CP samples order of cerebrospinal fluid homeostasis. Data are scarce were dissected from the lateral ventricles and AG samples regarding the cell-specific distribution of somatostatin re- were dissected from the superior sagittal sinus. ceptors in normal human CP and AGs, the sites of cerebrospinal fluid production and egress. Results: We demonstrated the presence of both somatostatin receptor types 1 and 2 in all samples of normal human CP and AGs. Arch Ophthalmol. 2002;120:1540-1543

HE PRODUCTION and ab- The purpose of this study was to in- sorption of cerebrospinal vestigate the localization of SSTR1 and fluid (CSF) are complex SSTR2 in human CP and AGs by immu- and poorly understood nohistochemistry. These findings are phenomena.1 The choroid discussed in the context of abnormalities plexusT (CP) is known to be the site of CSF associated with IIH, a disorder of CSF production in various mammalian spe- homeostasis. cies, including humans. Recent work has demonstrated synthesis of numerous pep- METHODS tides and their receptors in CP.2 Peptide molecules and their receptors involved in Fifteen samples of CP and 12 samples of AGs the process of CSF absorption, which takes were obtained at autopsy from 15 patients who place in the arachnoid granulations (AGs), died with no signs of intracranial disease (age range, 52-81 years). Samples of CP were dis- are largely unknown. sected from lateral ventricles, and AG samples Somatostatin (SST) is a ubiqui- were dissected from superior sagittal sinus. Tis- tously distributed neuropeptide with an- sue samples were obtained and processed in ac- tisecretory and antiproliferative proper- cordance with tissue procurement protocols From the William H. Havener ties.3 Its biological activity is mediated by from The University of Iowa, Iowa City. Speci- Eye Center (Drs Katz, Klisovic, 5 distinct membrane-bound SST recep- mens were fixed in 4% buffered formalin, em- and Lubow) and the Arthur tors (SSTR1-5).4 These receptors have been bedded in paraffin, and serially cut on a mi- James Cancer Hospital discovered on diverse human and animal crotome at 4 µm. Tissue sections were placed (Dr Katz), The Ohio State tissues involved in the production and ab- on charged slides, deparaffinized in xylene, and University, Columbus; and sorption of extracellular fluid.5-10 Octreo- rehydrated. The slides were placed into pre- Departments of Pediatrics heated 10% sodium citrate (Antigen Retrieval (Dr O’Dorisio) and Pathology tide acetate, a long-acting SSTR2-specific Solution; Biogenics, Napa, Calif), micro- (Dr Lynch) and Holden analogue, has been used as an adjunctive waved for 10 minutes, and left in a sealed con- Comprehensive Cancer Center treatment for idiopathic intracranial hy- tainer for 15 minutes, then washed in phos- (Dr O’Dorisio), The University pertension (IIH) without understanding phate-buffered saline 3 times. Afterward, the of Iowa, Iowa City. of its mechanism or site of action.11 slides were washed in OptiMax Wash Buffer

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Figure 1. Intense somatostatin receptor type 1 immunoreactivity (A) and somatostatin receptor type 2 immunoreactivity (B) in the epithelial and vascular endothelial cells of choroid plexus villi (diaminobenzidine, original magnification ϫ400).

(Biogenics) and incubated in Power Block (Biogenics) for 10 served on slides where primary Ab was omitted or in the minutes. Expression of SSTR1 and SSTR2 was studied by im- presence of competing antigen (data not shown). There munohistochemistry. Newly developed antibodies raised against was no apparent difference in the intensity of immuno- N-terminal 57–amino acids of SSTR1 and N-terminal 45– 12 reactivity for either receptor subtype in CP or AGs in re- amino acids of SSTR2 were used. Preparation of rabbit poly- gard to patient age (within the age range noted). clonal SSTR1 antibody (Ab) and SSTR2 Ab as well as specific- ity testing were previously described.12,13 The slides were incubated overnight at 4°C with primary SSTR1 Ab and SSTR2 COMMENT Ab (1:1000 and 1:2000 dilution in 3% bovine serum albumin in phosphate-buffered saline, respectively). Sections were washed with phosphate-buffered saline 3 times and incubated for 1 hour The SSTR1-ir and SSTR2-ir were observed in all human at room temperature with peroxidase-labeled polyclonal anti– CP and AG samples. Strong SSTR1-ir and SSTR2-ir in CP rabbit Ab (DAKO A/S, Glostrup, Denmark). Immunohisto- are consistent with the previously published results by chemical binding was visualized by incubating the tissue sec- Thoss and colleagues.14 They detected binding of radio- tions with diaminobenzidine chromogen for up to 15 minutes. actively labeled (labeled with iodine 125 [125I]) SST ana- The slides were then washed, counterstained, and cover- logues, ie, [125I]Tyr3-octreotide (binds SSTR2 and SSTR5), slipped. Antigen competition was performed by preabsorbing [125I]CGP 23996 (binds SSTR1 and/or SSTR4), and SSTR1 Ab and SSTR2 Ab with corresponding truncated SSTR1 125 and SSTR2 proteins that were used as antigens in antigen prepa- [ I]LTT-SRIF-28 (binds all types of SSTRs) in human ration (for 3 hours at room temperature; final concentration CP. The SST analogues used in that study bind more than of peptide was 50 mg/mL). Concentrations of preabsorbed Abs 1 receptor subtype. Those results were suggestive of but used for immunohistochemistry were corrected to account for not conclusive for the presence of any specific SSTRs on dilution with blocking peptide. CP. Distribution of SSTRs was not studied previously on AGs in humans or any primate species. RESULTS The exact function of SST and its receptors in CSF homeostasis in humans is unknown at present; how- In all 15 CP samples, intense membrane and cytoplas- ever, the abundance of SSTRs on CP and AGs implies their mic SSTR1 and SSTR2 immunoreactivity (ir) was de- involvement in the processes of CSF production and ab- tected on all epithelial cells and vascular endothelial cells sorption. The CSF dynamics might be affected locally at in capillaries within individual villi (Figure 1). Equally the level of CP and AGs through SSTRs or systemically strong SSTR1-ir and SSTR2-ir was observed in arach- by modulating the levels or activity of circulating hor- noid cells, connective tissue, and dural layer cells in all mones (growth hormone, insulinlike growth factor [IGF] AG samples (Figure 2). No immunostaining was ob- 1, insulin, leptin, etc). In addition, SSTR1 and SSTR2

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Figure 2. Strong somatostatin receptor type 1 immunoreactivity (A) and somatostatin receptor type 2 immunoreactivity (B) on arachnoid cells, connective tissue, and dural layer cells in arachnoid granulations. Note red blood cells within the superior sagittal sinus (A) (diaminobenzidine, original magnification ϫ200 [A] and ϫ400 [B]).

might modulate blood flow in CP capillaries and filtration of circulating levels of IGF-1.15 This is of special in- tion in CP epithelium. terest, because it is now apparent that a side effect of The SST axis has a role in ion transport, extracellu- recombinant IGF-1 treatment in children with growth lar fluid production, and absorption in different mamma- hormone receptor deficiency is a transient increase in in- lian and human tissues.3 Recent work has demonstrated tracranial pressure.16-26 The increased intracranial pres- SST effects in kidney and in the eye. Intravenous injec- sure resolves with complete cessation of IGF-1 treat- tion of SST modulates glomerular filtration rate, renal ment or reduction of IGF-1 dose by 50%.25 In this regard, plasma flow, urine volume, and water clearance in nor- it is important to recognize that CP epithelium has one mal human kidney.5,7 The recent work of Balster and col- of the highest densities of IGF-1 receptors in the human leagues10 demonstrated SSTR1 and SSTR2 gene expres- brain.27,28 These data suggest a role for high plasma IGF-1 sion as well as orderly segmental distribution of SSTR1-ir levels in the pathogenesis of increased intracranial pres- and SSTR2-ir along the glomerular and tubular system in sure in children. normal human kidney tissue.10 Such data offer us a rea- The IGF-1 axis also has a significant role in the patho- sonable theoretical model for a similar role of the SST axis genesis of obesity. Obese patients often have numerous in CSF dynamics. hormonal abnormalities, including glucose intolerance, In the rabbit eye, SST inhibits aqueous production insulin resistance, hyperinsulinemia, low plasma levels by stimulating adenylyl cyclase activity and release of in- of growth hormone, and high levels of free IGF-1 due to tracellular calcium in nonpigmented epithelium of cili- overproduction of IGF-1 by adipocytes.29-31 Idiopathic in- ary processes.6,8,9 Using reverse transcription polymerase tracranial hypertension is a disease that predominantly chain reaction, Klisovic and colleagues13 recently de- affects obese women of childbearing age. Octreotide treat- tected gene expression for SSTR1, SSTR2, and SSTR4 in ment in such patients might decrease levels of circulat- ciliary body in normal human eyes. In addition, SSTR1-ir ing IGF-1 but could also counteract its biological activ- and SSTR2-ir were detected on nonpigmented epithe- ity at the level of CP epithelium. In human retinal pigment lium of ciliary processes, in marginal capillaries in ciliary epithelial cells (SSTR1 and SSTR2 positive), SST and oc- processes, and on endothelial cells in trabecular mesh- treotide inhibit biological effects of IGF-1 by inhibition work. These data again suggest an analogous role for SST of autophosphorylation of IGF-1 receptors, thereby in- in aqueous dynamics of the eye, as well as the CSF. hibiting the IGF-1 signaling cascade.32 The best studied activity of SST and its analogues In conclusion, we demonstrated the presence of in humans is the inhibition of the release of growth hor- SSTR1 and SSTR2 in normal human CP and AGs. Analo- mone from pituitary, which in turn results in the reduc- gous to their demonstration and to their function in kid-

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©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 ney and ocular tissues, these receptors may be involved 13. Klisovic DD, O’Dorisio MS, Katz SE, et al. Somatostatin receptor gene expres- in the processes of CSF production and absorption in hu- sion in human ocular tissues: RT-PCR and immunohistochemical study. Invest Ophthalmol Vis Sci. 2001;42:2193-2201. mans and may play a role in the increased intracranial 14. Thoss VS, Piwko C, Probst A, Hoyer D. Autoradiographic analysis of somato- pressure in IIH. statin SRIF1 and SRIF2 receptors in the human brain and pituitary. Naunyn Schmiedebergs Arch Pharmacol. 1997;355:168-176. Submitted for publication December 28, 2001; final revi- 15. Brazeau P, Vale W, Burgus R, et al. Hypothalamic polypeptide that inhibits the se- sion received June 10, 2002; accepted June 13, 2002. cretion of immunoreactive pituitary growth hormone. Science. 1973;179:77-79. 16. 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