Introduction

The Endocannabinoid System (ECBS) is formed by lipid ligands (endocannabinoids), the enzymatic machinery for their synthesis and degradation and their specific G-protein coupled CB1 and CB2 receptors. The most important endocannabinoids are 2-arachydonoylglycerol (2-AG) and anandamide (AEA)1. These compounds are involved in the control of neural stem cell biology2 and many of their effects are mediated by the cannabinoid receptor CB1. CB1 receptor is expressed in all neurogenic niches in rodents, including the ependymal region of the spinal cord (reviewed in3). In this region, that holds neural stem cell potential4,5, a subpopulation of cells expresses high levels of CB1 receptor (CB1HIGH cells) and proliferate after lesion or during postnatal development in rats6. However, the ependymal region of the adult human spinal cord is strikingly different from that of rodents and other primates: although it contains ependymal cells, lacks a patent central canal and shows perivascular pseudorosettes7,8,9. This means that observations made in rodents should be validated in human tissue to understand the composition and the regenerative potential of this niche. Here we have explored the presence of the ECBS and searched for an equivalent of rat and mice CB1HIGH cells in adult human spinal cord.

Results and Discussion

We found that human ependymal region consistently expresses CB1 cannabinoid receptor (CNR1 gene; Table 1). CB1 receptor could be the target of locally produced 2-AG, since we also found expression (although non enrichment) of enzymes related with 2-AG synthesis and degradation: diacylglycerol lipase α (DAGLA), diacylglycerol lipase β (DAGLB), monoacylglycerol lipase (MGLL) and abhydrolase domain-containing proteins – 6 (ABHD6) and –12 (ABHD12). On the contrary, we could not find consistent expression of enzymes related with direct anandamide synthesis or degradation (NAPE-phospholipase D and fatty acid amide hydrolase, respectively). However, it should be noted that alternative enzymatic routes have been described for AEA, involving glycerophosphodiester phosphodiesterase and N-acylethalnolamine-hydrolyzing acid amidase that have been not tested here2. We also did not find expression of CB2 cannabinoid receptor or the related GPR55 receptor. In previous works, we observed expression (but not enrichment) of PPAR-α, another cannabinoid-related receptor1, in human ependymal region9.

Table 1 Relative expression of endocannabinoid system related genes in the adult human ependymal region compared with ventral horn.
Full size table

When compared with ventral horn, only CNR1 (CB1 receptor) was significantly enriched at the ependymal region (Table 1). Accordingly, we found a strong CB1 immunoreactivity in central gray matter by immunohistochemistry (Fig. 1B–J). But CB1 enrichment in adult humans ependyma is not equivalent to that found in rodents: In humans, CB1 is expressed by astrocytes, forming part of the gliosis that accompanies central canal closure (Fig. 1C–E) and in the GFAP+ hypocellular ribbon of perivascular pseudorosettes (Fig. 1F–K)9,10. CB1 receptor is also expressed in astrocytes from other spinal cord areas (Fig. 2) and its intensity is apparently related to high GFAP expression. Accordingly, a strong CB1 expression has been reported in reactive astrocytes of human pathologies like spinocerebellar ataxia11 or temporal lobe epilepsia12. The role of astrocytic CB1 could be multiple: protection13, metabolism increase14, control of inflammation15,16,17, inhibition of glutamate transporters18 or release of neurotransmitters such as glutamate19, ATP and D-serine20.

Figure 1
figure 1

CB1 cannabinoid receptor in adult human spinal cord.

(A) Myelin staining of a representative thoracic spinal cord section. Square depicts the area shown in (C–E). (B) In low magnification a strong CB1 immunoreactivity can be found in dorsal horn, lamina X and ventral gray matter. (C–E) Higher magnification of central gray shows CB1 expression in GFAP+ areas surrounding the Vimentin+ cells at the ependymary region (EpR). Square highlights the location of a perivascular pseudorosette (PvPR). (F–J) Strong CB1 immunoreactivity is found at the GFAP+ domains around and inside perivascular pseudorosettes, including the GFAP ribbon at the hypocellular region of the pseudorosette (hcr, outlined in white). In PvPRs cells are arranged around a central vessel ((I) arrow). (K) Quantification supports qualitative observations: CB1 immunoreactivity is significantly accumulated in GFAP+ areas. (L) Detail of the dorsal aspect of an ependymal region with a patent central canal. Square depicts location of images M and N. (M) CB1HIGH ependymal cells (empty arrows) can be found intermingled with ependymocytes lining the central canal. GFAP+ cells contacting central canal lumen (arrowhead) are CB1−. (N) Detail of M, showing a CB1HIGH cell with a dim staining of GFAP in the apical region. ***T student, p < 0.001; CC, Central Canal; EpR, ependymal region; hcr, hypocellular region; PvPR, perivascular pseudorosette; WM, white matter. Scale bars: A,B = 1 mm; C-E: 100 μm; F-L = 50 μm; M,N = 25 μm.

Full size image
Figure 2
figure 2

CB1 immunoreactivity can be found in astrocytes of other regions in the spinal cord.

(A–F) CB1 is expressed in the processes of GFAP+ and Vim+ astrocytes (arrows) at the dorsolateral white matter. (G–I) A strong CB1 expression can be found in astrocytic processes at the subpial region. Scale bars: 25 μm.

Full size image

Interestingly, we obtained some sections from adult individuals in which parts of the central canal were patent. In those sections, we found ependymal cells with high expression of CB1 receptors lining the canal (Fig. 1L–N), resembling those CB1HIGH cells described for rats and mice6. These cells were mostly GFAP-, except for a very dim expression at the apical pole (Fig. 1N), in contrast with strongly GFAP+ cells embeded in the ependymal layer (Fig. 1M).

Our results support the existence of ependymal CB1HIGH cells across species and may encourage further studies on this subpopulation, although only in cases when there is central canal patency, i.e. childhood and upper cervical levels8,9. But in the majority of adult ependyma, CB1 is enriched in astrocyte domains and cannabinoids may play a different role, that still might be relevant, in terms of homeostasis maintenance and response to injury.

Methods

Human tissue was obtained from the HUFA BioBank (Alcorcon, Spain) and the HUB-ICO-IDIBELL BioBank (Hospitalet de Llobregat, Spain). Samples were obtained from donor individuals deceased without clinical or histopathological involvement of the spinal cord (Table 2). Donation always included a written informed consent from donors while alive or from their families after death. Data from donors and handling of samples were carried out after approval by the Clinical Research Ethical Committee (CEIC) in Toledo (Spain), in accordance with the Spanish law and International Guidelines (LOPD 15/1999; RD 1720/2007; Helsinki declaration, 2008).

Table 2 Postmortem Spinal Cord tissue samples used for immunohistochemistry (IHC) and/or Laser Capture Microdissection (LCMD).
Full size table

Gene expression in human ependymal region

All procedures were performed according to our published protocol9. Briefly, fresh frozen spinal cord blocks were cut in 25 μm thick sections and the ependymal region microdissected with a Laser Dissection Microscope. RNA extraction, amplification and reverse transcription were performed as previously described9. We also collected microdissected portions of ventral horn, which we used as a non-neurogenic, non-ependymal reference for gene expression.

Gene expression was studied with Taqman PCR Assays (Life Technologies, Madrid, Spain) either incorporated in Taqman Low Density Arrays (DAGLA, #Hs00391374_m1; DAGLB, #Hs00373700_m1; MGLL, #Hs00200752_m1; NAPEPLD, #Hs00419593_m1) or in individual assays (ABHD6, #Hs00977889_m1; ABHD12, #Hs01018047_m1; CNR1, #Hs01038522_s1; CNR2, #Hs00361490_m1; FAAH, #Hs01038660_m1; GPR55, #Hs00271662_s1). We used 18S gene as an endogenous control (18S, #Hs03003631_g1). For assays incorporated on TLDAs, we added 1.25 ng cDNA/well. For assays performed individually, we added 1.5 ng cDNA/well. Assays were run on an Applied Biosystems® 7900HT Fast Real-Time PCR System. Data were analysed as described9 using automatic detection of Ct, normalized with the endogenous gene (ΔCt vs 18S). Only genes expressed in at least three out of four samples were considered as consistently expressed and included in statistics. Enrichment was defined as higher and statistically significant expression in ependymal region vs ventral horn (Student’s t-test with ΔCts, p < 0.05). To obtain folds of enrichment, we used Relative quantity formula, RQ = 2^−ΔΔCt.

Immunohistochemistry

To improve signal to noise ratio and avoid autofluorescence, we amplified CB1 immunoreactivity using Tyramide Signal Amplification System (TSA Plus Cyanine 3 System #NEL744001KT, Perkin Elmer, USA). Free floating vibratome sections (40 μm) were rinsed on 0.1 M phosphate-buffered saline containing 0.5% bovine serum albumin +0.3% Triton X-100. Endogenous peroxidase inhibition and antigen demasking were performed as described9. Sections were then blocked with TSA Blocking Solution (45′) and incubated for 2 days with primary antibodies diluted in rinse solution +10% Normal Donkey Serum: guinea pig anti-CB1 (1:2000, #CB1-GP-Af530-1, FSI, Japan), rabbit anti-GFAP (1:2000, #Z0334, DAKO, Spain) and mouse anti-Vimentin (1:300, #M0725, DAKO, Spain). Immunoreactivity was visualized by incubating sections with Alexa 488-, Alexa 555- and Alexa 633- secondary antibodies (1:1000, Invitrogen, Spain) or horseradish peroxidase donkey anti-guinea pig antibody (1:300, Jackson Immunoresearch, UK) followed by Tyramide-Cy3 diluted in TSA Amplification Buffer (1:50). Samples were analyzed with a LEICA SP5 confocal microscope. We ruled out the interference of nonspecific staining by omitting primary antibodies. We set the confocal parameters at a point where no signal was observed in these primary antibody controls and those settings were used for all the image acquisitions (Supplementary Figure 1A–F). Furthermore, as discussed in several reports, there is a variety of antibodies against CB1 receptor and some of them may show non-specific staining21,22,23. The specificity of CB1 antibody used for this report has been extensively validated by other laboratories and ourselves in previous works6,24,25. We show here an additional validation in the Supplementary Figure 1 by using immunohistochemistry and TSA amplification on wild type (C57BL/6N) and CB1 knockout mice tissue (kindly donated by Dr. Galve-Roperh26). Using restrictive confocal parameters (as we did for humans), we got rid out of autofluorescence, background staining and most of the non-specific staining observed in the knockout mice that, in these conditions, is limited to a dim intracellular neuronal staining, largely different from that observed in the wild type mice (Supplementary Figure 1L-Q). All post-capture image modifications were identically performed for controls, including cropping, noise reduction and minor adjustments to optimize contrast and brightness.

To quantitatively support CB1 enrichment in the astrocytic area, we calculated the fraction of CB1 found in GFAP+ vs GFAP− areas on confocal planes (image size 190 μm × 190 μm) using Fiji (http://pacific.mpi-cbg.de). For this, we outlined GFAP borders using manual Threshold with Otsu Filter and used this ROI on the CB1 image corresponding to the same confocal plane. We measured CB1+ Area inside and outside the selection (GFAP+ and GFAP− areas, respectively) and expressed them as % of total CB1 staining (Fig. 1K). We used Student T-test for statistical comparisons.

Additional Information

How to cite this article: Paniagua-Torija, B. et al. CB1 cannabinoid receptor enrichment in the ependymal region of the adult human spinal cord. Sci. Rep. 5, 17745; doi: 10.1038/srep17745 (2015).