J Mol Neurosci (2012) 46:450–458 DOI 10.1007/s12031-011-9613-4
Inflammation-Induced Changes in the Chemical Coding Pattern of Colon-Projecting Neurons in the Inferior Mesenteric Ganglia of the Pig Joanna Wojtkiewicz & Maciej Równiak & Robert Crayton & Monika Barczewska & Marek Bladowski & Anna Robak & Zenon Pidsudko & Mariusz Majewski
Received: 21 April 2011 / Accepted: 25 July 2011 / Published online: 9 August 2011 # Springer Science+Business Media, LLC 2011
Abstract The present study examines the chemical coding of the inferior mesenteric ganglia after chemically induced colitis in the pig animal model. In all animals (n=6), a median laparotomy was performed under anesthesia, and the Fast Blue retrograde tracer was injected into the descending colon wall. In experimental animals (n=3), the thick descending colon were injected with formalin solution to induce inflammation. The animals were euthanized and the inferior mesenteric ganglion was harvested and proElectronic supplementary material The online version of this article (doi:10.1007/s12031-011-9613-4) contains supplementary material, which is available to authorized users. J. Wojtkiewicz (*) : M. Barczewska Department of Neurology and Neurosurgery, Division of Neurosurgery, Faculty of Medical Sciences, University of Warmia and Mazury, Warszawska 30, 10-082 Olsztyn, Poland e-mail:
[email protected] M. Równiak : A. Robak Department of Comparative Anatomy, Faculty of Biology, University of Warmia and Mazury, Olsztyn, Poland R. Crayton Department and Clinic of Urology, Faculty of Medical Sciences, Medical University of Warsaw, Warsaw, Poland M. Bladowski : M. Majewski Department of Human Physiology, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland Z. Pidsudko Department of Animal Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury, Olsztyn, Poland
cessed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P (SP), vesicular acetylcholine transporter (VAChT), or galanin (GAL). Immunohistochemistry revealed significant changes in the chemical coding pattern of inferior mesenteric ganglion neurons. In control animals, Fast Bluepositive neurons were immunoreactive to TH, NPY, SOM, VIP, LENK, CB, and NOS. In the experimental group, TH, NPY, SOM, VIP, and LENK expressing neurons were reduced, whereas the number of neurons immunoreactive to CB, NOS, and GAL were increased. The increase of socalled neuroprotective neuropeptides suggests that the changes in the chemical coding of inferior mesenteric ganglion neurons reflect adaption under pathological conditions to promote their own survival. Keywords Inflammation . Neuropeptides . Colon supplying neurons . Inferior mesenteric ganglion . Pig
Introduction Colitis is an inflammatory condition of the large bowel which is characterized by multiple etiology. These include infections (Sherman et al. 2010), poor blood supply (Papa et al. 2008; Carlson and Madoff 2011), autoimmune reactions (Hibi et al. 2002; Khan et al. 2011), and chemical irritation (Gonkowski and Calka 2010). Each particular etiology produces its own and unique pattern of pathological changes, but they all have common factors that lead to colon damage (Sanchez-Munoz et al. 2008). For example, the common symptoms of ulcerative colitis include rectal
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bleeding and diarrhea (Wouters and Boeckxstaens 2011). Chemically induced colitis can occur as a result of enema administration containing various chemicals irritants (Hsu et al. 2009; Desai and Orledge 2010). Most cases have occurred after accidental contamination from endoscopes with glutaraldehyde and/or hydrogen peroxide or through the unintentional administration of caustic chemicals (Stein et al. 2001; Hsu et al. 2009; Desai and Orledge 2010). Intentional administration of corrosive enemas has been implicated in sexual practices, bowel cleansing, or in suicide attempts. In spite of the different etiology, patients with ulcerative or chemically induced colitis both present with similar nonspecific symptoms that include abdominal pain, rectal bleeding, and/or diarrhea. Since various chemicals can induce similar symptoms of and pathologies of colitis, these agents could be employed for studying colon inflammation in laboratory conditions. There are several models of colon inflammation based on various chemicals used to induce such phenomenon (for review, see Elson et al. 1995). These models are a valuable tool for discovering subtle details concerning inflammatory processes and their influences on various cell types (Lomax et al. 2005, 2006). They are also especially useful for studying the influences of inflammation on the enteric nervous system (Vasina et al. 2006). For example, Gonkowski et al. (2010) described the impact of chemically induced colitis on the somatostatin immunoreactivity in the colon. According to these authors, subpopulation of somatostatincontaining neurons was three times greater in the inner submucosal plexus in inflamed animals in comparison with healthy controls, whereas the number of somatostatinimmunoreactive nerve fibers in the mucosal layer was considerably reduced (Sienkiewicz et al. 2004). Gonkowski et al. (2010) described the decrease in the number SOM, SP, CGRP-positive nerve fibers in mucosal plexuses. Previous studies revealed morphological changes in the enteric nerve system associated with loss of myenteric plexus neurons (Boyer et al. 2005; Wedel et al. 2010). It is well-known that neurons show a high degree of plasticity in response to pathological situations (Sharkey and Kroese 2001; Ekblad and Bauer 2004). Such adaptive changes usually induce both the up and downregulation of neurotransmitter expression to help neurons to survive under pathological conditions (Sharkey and Kroese 2001; Ekblad and Bauer 2004). The selective up and downregulation of expression of various neurotransmitters was also described in animals afflicted with proliferative enteritis (Pidsudko et al. 2008a, 2008b) and swine dysentery (Sienkiewicz et al. 2004). In the present study, colon inflammation was induced by the administration of 10% formaldehyde to the wall of the descending colon in experimental pig animal models according to previously described methodology (Gonkowski and
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Calka 2010). In such laboratory models, the influence of colitis on the colon-supplying neurons located in the inferior mesenteric ganglion (IMG) of the pig is analyzed using the combined retrograde tract-tracing method and doublelabeling immunohistochemistry. The pig was employed as an experimental animal model as its physiological features are close to those of the human and the results provide a basis for clinical studies of medical conditions. These data suggest that changes in chemical coding of IMG neurons induced by colitis may have an important role in the adaptation of IMG local nerve circuits under pathological conditions and their survival.
Material and Methods Study Subjects The study was performed on six immature female pigs of the Large White Polish breed (approximately 8 weeks old) divided into two groups: control animals (C group; n=3) and animals with chemically induced colitis (INF group; n=3). All animals were housed and treated in accordance with rules approved by the ethics committee (conforming to Principles of Laboratory Animal Care, NIH publication no. 86–23, revised 1985). Anesthesia and Surgery Surgery was performed under fractionated thiobarbital (Thiopenthal, Sandoz, Austria; 20 mg/kg body weight, i.v.) anesthesia. Prior to administration (30 min) of the main anesthetic, the animals were pretreated with atropine sulfate (Polfa, Poland; 0.04 mg/kg body weight, s.c.), and azaperone (Stressnil, Janssen Pharmaceutica, Belgium; 2.0 mg/kg body weight, i.m.). All animals (n=6) were laparotomised and injected with 5% aqueous solution of the fluorescence retrograde neuronal tracer Fast Blue (FB; Dr. K. Illing GmbH, Gross-Umstadt, Germany) into the wall of the descending colon. A total volume of 40 μl 5% aqueous dye solution was injected into the exposed descending colon using a Hamilton syringe with a 26-gauge needle. Multiple (n=40; 1 μl each) injections were made into the right part of descending colon. Three weeks later, the animals of the INF group underwent a median laparotomy to expose the descending colon. An injection with 80 μl of 10% formalin solution (microinjection of 5–8 μl) into the wall of the descending colon was done as described previously by Gonkowski et al. (2010). The chemically induced colitis was then confirmed by both the gross-anatomical and macroscopic examination of the bowel wall, as descending in details by Gonkowski and Calka (2010) and Gonkowski et al. (2010). After 7 days, all animals (C and INF) were
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re-anesthetized and euthanized by an overdose of thiobarbital and perfused transcardially with 4% buffered paraformaldehyde (pH 7.4). Following perfusion, small tissue blocks comprising of the IMG were collected from all studied animals and postfixed by immersion in the same fixative for 4 h, washed twice in 0.1 M phosphate buffer (pH=7.4, 4°C), and then stored in 18% sucrose at 4°C until sectioning. Immunofluorescence experiments Ten-micrometer thick cryostat sections of IMGs were processed for routine double-immunofluorescence labeling using primary antisera raised in different species and species-specific secondary antibodies (Table 1). All sections were incubated in humid chambers with a mixture of two primary antisera raised in different species (overnight). The mixture consisted of antibodies directed towards calbindin (CB) and antisera raised against tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), leucine-enkephalin (LENK), substance P (SP), vesicular acetylcholine transporter (VAChT), or galanin (GAL) (Table 1). Then, sections were incubated with a mixture of FITC-conjugated secondary antisera and biotin (1 h). The latter antibodies were finally visualized by additional incubation of sections with streptavidin–CY3 complex (1 h). After staining, sections were mounted with carbonate-buffered glycerol (pH 8.6) and cover-slipped. Each step of immunolabeling was followed by rinsing of the sections with PBS (3×15 min). Table 1 Specification of immunoreagents
Controls Standard controls, i.e., preabsorption for the neuropeptide antisera (20 mg of appropriate antigen per 1 ml of corresponding antibody at working dilution) and the omission and replacement of all primary antisera by non-immune sera or PBS were applied to test both antibody and method specificity. Counts and statistics The sections were viewed under a Olympus BX51 fluorescence microscope equipped with a barrier filter for FB and the respective filters for FITC and CY3. Microphotographs were acquired with a CCD camera connected by a PC equipped with Olympus image analysis software (ver. 3.2; Soft Imaging System GmbH, Münster, Germany). To determine the relative number of FB-positive cells, the neurons were counted in every 16th section in whole ganglion to avoid double counting. At least 560 FB+ neurons were analyzed for any combination of antigens (see Table 2). Only neurons with clearly visible nuclei were counted. Data pooled from two different animal groups were expressed as means±standard error of mean (SEM) and were analyzed by using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA). Significant differences between groups of data were evaluated with the use of Student's t tests. (*P≤ 0.05, **P≤0.01 and ***P≤0.001).
Results The porcine IMG in control animals was found to contain many retrogradely labeled neurons projecting to the descend-
Antigen
Code
Host Species
Dilution
Supplier
Primary antibodies CB (CB-Dk28)
CB-38
Rabbit
1:20,000
SWANT
MAB 318 NZ1115 8330-0009 9535-0504
Mouse Rat Rat Mouse
1:80 1:300 1:100 1:2,000
Chemicon Biomol Biogenesis Biogenesis
1:2,000 1:1,000 1:300 1:2,000 1:1,000
Sigma Biogenesis Biogenesis Phoenix Peninsula
1:800 1:800 1:1,000 1:1,000 1:1,000 1:1,000 1:9,000
Jackson Jackson Jackson Jackson DAKO BioTrend Jackson
TH NPY SOM VIP
NOS N2280 Mouse LENK 4140-0355 Mouse SP 8450-0505 Rat VAChT H-V007 Goat GAL T-5036 Guinea pig Secondary reagents Donkey anti-mouse IgG (H+L) conjugated with FITC Donkey anti-rat IgG (H+L) conjugated with FITC Donkey anti-guinea pig IgG (H+L) conjugated with FITC Donkey anti-goat IgG (H+L) conjugated with FITC Biotinylated goat anti-rabbit immunoglobulins Biotin conjug. F(ab)′ fragm. of affinity purified anti-rabbit IgG (H+L) CY3-conjugated Streptavidin
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Table 2 Percentages of the main subsets of retrograde-labeled neurons in the inferior mesenteric ganglia of control (C) and inflamed (INF) animals FB+/CB+/P−
FB+/CB+/P+ C
INF
TH NPY
32.3±4.8 25.0±5.2
19.3±3.8 2.7±1.4
SOM
12.8±4.5
VIP NOS
1.1±1.0 0
LENK
C
FB+/CB−/P+
FB+/CB−/P−
INF
C
INF
C
INF
2.4±1.2 4.1±2.8
20.9±3.1 45.5±4.3
56.4±4.2 59.3±3.7
13.2±6.2 1.2±0.4
6.9±1.2 11.4±2.5
46.7±3.2 50.6±2.5
1.0±0.15
19.2±2.8
48.0±2.3
5.01±3.2
0.1±0.1
62.5±3.6
51.1±2.4
0.2±0.2 4.3±1.6
35.6±3.7 37.0±2.5
43.8±1.2 33.0±0.3
3.1±0.8 1.5±1.3
0.2±0.1 2.2±1.5
63.4±1.9 63.5±1.9
55.9±0.7 60.5±2.1
0
1.3±1.3
32.7±1.9
42.7±2.6
1.8±1.1
0.13±0.1
65.2±3.2
55.9 ±3.6
SP VAChT
0 0
0 0
34.7±2.5 38.3±1.8
49.5±0.5 51.9±0.6
0 0
0 0
65.5±2.5 61.7±1.8
50.5±0.5 48.1±0.6
GAL
0
0.2±0.1
39.3±1.8
50.0±3.0
0
0.3±0.2
60.7±1.8
49.4±2.4
Data were expressed an mean±standard error of mean (SEM) P peptide
ing colon (FB+); these were distributed bilaterally, i.e., within both the left and right ganglia. In each of these ganglia, FB+ cells formed a neurochemically heterogenous population composed of neurons using various substances as their main neurotransmitters (Fig. 1 and Table 2). The great majority of FB+ cells (80–90%) used TH or NPY as the main neurotransmitters (Fig. 1b and Table 2). Another large subpopulation of FB+ neurons contained CB. These cells constituted 30–40% of FB+ neurons (Fig. 1a and Table 2). In addition, small subpopulations of FB+ neurons co-expressed SOM (16–18%), VIP (4–5%), NOS (1–2%), and LENK (1–2%) (Fig. 1c–f and Table 2). No FB+ perikarya was immunopositive to SP, VAChT, and GAL (Table 2). Moreover, each of these subpopulations of FB+ neurons is also neurochemically heterogeneous. For example, around 32% of FB+/CB+ cells co-express TH. More than 25% of them contain NPY. Neurons expressing CB and SOM or VIP constitute 12% and 1% of FB+/CB+ cells, respectively (Fig. 1c–d and Table 2). On the other hand, none of the FB+/CB-positive perikarya was immunopositive to NOS, LENK SP, VAChT, and GAL (Table 2). In animals with chemically induced colitis, the size of the whole population of FB+ neurons did not change; however, the coding patterns of many FB+ cells changed (Fig. 1a′–e′ and Table 2). For example, FB+/TH+, FB+/ NPY+, FB+/SOM+, and FB+/VIP+ neurons were more or less reduced (Fig. 1b′–d′, Table 2; Fig. 2). The population of FB+/TH+ cells in colitis suffering pigs is almost three times smaller in comparison with control animals (***P≤0.001) (Fig. 1b′; Table 2; Fig. 2). Reduction among FB/NPY (***P≤0.001), FB/SOM (**P≤0.01), and FB/VIP (**P≤ 0.01) neurons is also statistically significant (Fig. 1b′–d′ and Table 2; Fig. 2). On the other hand, FB+/CB+ (**P≤ 0.01), FB+/NOS+ (**P≤0.01), and FB+/GAL+ (*P≤0.05) neurons increase in number in sick animals. (Fig. 1b′, e′ and
Table 2; Fig. 2). Cells containing galanin (FB+/GAL+) were observed only in inflamed animals (Table 2). Changes in the chemical coding pattern of FB+/CB+neurons subpopulation were quite similar to these, concerning the whole FB+ cells population. For instance, strong reduction was observed among FB+/CB+/TH+ (*P≤0.05), FB+/CB+/NPY+ (**P≤ 0.01), FB+/CB+/SOM+ (**P≤0.01), and FB+/CB+/VIP+ (*P≤0.05) neurons (Fig. 1b′–d′ and Table 2; Fig. 2). On the other hand, FB+/CB+/NOS+ (**P≤0.01), FB+/CB+/ LENK+ (P>0.5), and FB+/CB+/GAL+ (*P≤0.05) neurons increase in number in sick animals (Table 2; Fig. 2).
Discussion Results of the present study indicate that although chemically induced colitis had no influence on the number of colon-projecting IMG neurons, it had a large impact on their chemical coding pattern. Thus, while the numbers of FB+/TH+, FB+/NPY+, FB+/SOM+, and FB+/VIP+ neurons were strongly reduced in animals with chemically induced colitis, FB+/CB+, FB+/NOS+, and FB+/GAL+ neurons increased in number in these animals. It has been shown that TH + , NPY +, and SOM + cells are involved in controlling and modulating the motility, blood flow, and gland secretion in the colon and these neurons form the majority of colon-projecting IMGs (Gershon and Sherman 1982; Landberg et al. 1984; Costa and Furnesss; 1984; Gershon and Sherman 1987; Tassicker et al. 1999). Thus, TH+ and SOM+ neurons were thought to modulate colon motility because their fibers and terminals were found around myenteric ganglion neurons, which are essential for enteric reflex motor activity. TH+ terminals form basket-like endings around cholinergic motor neurons and 5-HT handling neurons (Gershon and Sherman 1982, 1987;
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Fig. 1 Representative images of colon-projecting neurons located in IMG of the pig. All images are composites of merged images taken separately from the blue (Fast Blue), red, and green fluorescent channels. a–f Control animals (C), a′–f′ animals with chemically induced colitis (INF). Scale bar=50 μm. a FB+ neurons labeled to TH (green) and CB (red) immunonegative (small arrows), a′ FB+ neurons labeled to CB (red) and TH (green) immunonegative (three single arrows); FB+ neuron labeled to TH (green) and CB (red) immunonegative (small arrow); FB+ neuron labeled to TH (green) and CB (red) (double arrow); b FB+ neuron labeled to TH (green) and CB (red) (double arrow), b′ FB+ neuron labeled to NPY (green) and CB
(red) immunonegative (small arrow); c FB+ neurons labeled to CB (red) and SOM (green) (three double arrows), c′ FB+ neuron labeled to CB (red) and SOM (green) (double arrow); d FB+ neuron labeled to CB (red) and VIP (green) immunonegative (single arrow) and FB+ neuron labeled to VIP (green) and CB (red) immunonegative (small arrow), d′ FB+ neuron labeled to CB (red) and VIP (green) immunonegative (single arrow); e A group of NOS+ (green) neurons unlabeled to FB and CB, e′ FB+neuron double-labeled to NOS (green) and CB (red) (small arrow); f FB+ neurons labeled to CB (red) and LENK (green) immunonegative (two single arrows), f′ FB+ (blue) neurons unlabeled to CB and LENK
Tassicker et al. 1999). Noradrenaline was shown to acts presynaptically to inhibit the release of neurotransmitters in the myenteric plexus (Paton and Vizi 1969; Manber and Gershon 1979; Furness et al. 1990; Tassicker et al. 1999). NPY-containing neurons of the IMG were considered vasomotor neurons because their fibers and terminals surrounded arterial vessels in the colon wall (Landberg et al. 1984; Costa and Furnesss 1984), thus, influencing their
control on arterial blood flow though the colon wall. Apart of being involved in the neurogenic control of intestinal motility and blood flow through the organ, SOM, NPY, and VIP were also postulated to play other important roles during inflammatory processes of the bowel. Although SOM, NPY, and VIP were suggested to be mediators of anti-inflammatory reactions, the neurons releasing them were thought to be involved in colon protection during
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Fig. 2 Diagram illustrating the FB+cell numbers which were immunoreactive to appropriate antigens in IMGs of control animals (dark grey bars) and animals with chemically induced colitis (light grey bars) [percent]. Student's t test, ***P≤0.001; **P≤0.01; *P≤0.05
inflammation (Reubi et al. 1994). However, it appears that when the porcine colon is challenged by a chemical irritant, they are involved in other mechanisms as the present data suggests that neurons with co-localized SOM+, NPY+, and VIP+ neurons were strongly reduced in the IMG of animals with chemically induced colitis. SOM has been considered to be anti-inflammatory and anti-nociceptive factor which may be actively involved in the pathophysiology of bowel inflammation (Reubi et al. 1994), reducing nociceptor excitation, and sensitization during inflammation (Hasler et al. 1993; Plourde et al. 1993; Abd El-Aleem et al. 2005; Pinter et al. 2006). This could be indirectly supported by findings of Pidsudko et al. (2003), who also have reported
an increase in the number of SOM+ neurons in dorsal root ganglion of pig with chemically induced ileitis. NPY has been implicated as mediator involved in the pathogenesis of numerous gastrointestinal disorders, including malabsorption, short gut, inflammatory bowel disease, and various forms of pancreatitis (Vona-Davis and McFadden 2007). However, the exact physiological role of NPY in all these pathologies is not yet clear and requires further investigation. The strong reduction in the number of NPY+ neurons observed in the present study is of interest as the number of such coded neurons were studied so far in the pig in various enteric diseases are usually elevated (Majewski et al. 2004; Czaja et al. 2005;Lakomy et al. 2005). For example, it has
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been shown that proliferative enteropathy evoked an increase in the number of NPY+ IMG neurons supplying descending colon (Majewski et al. 2004). Similarly, in animals with porcine dysentery, NPY expression has been reported to be elevated in the IMG (Lakomy et al. 2005). As VIP is widely accepted as an anti-inflammatory factor (Delgado and Ganea 2001; Delgado et al. 2002; Abad et al. 2003), an increase of the number of VIP-expressing cells could be expected during chemically induced colitis. Moreover, in the present study, the number of VIPexpressing IMG neurons was found to be reduced in animals with chemically induced colitis. In contrast to NPY, SOM, or VIP-expressing IMG neurons, in the present study we have observed that perikarya containing CB, NOS, or GAL become more numerous in animals with chemically induced colitis. Interestingly, all these neurotransmitters were shown to be neuroprotective in nature and, when released, were able to support challenged neurons to survive (Hugon et al. 1996). Calbindin D28K is a calcium-binding protein, which is thought to have a neuroprotective action against various cellular insults (Hugon et al. 1996) as well as calciummediated neurotoxicity (Iacopino et al. 1992). It is not surprising that under pathological conditions, affected neurons reacted with the upregulation in its synthesis. The upregulation of CB in neurons was observed in various pathological conditions (Pfannkuche et al. 2008; Sojka et al. 2010). Nitric oxide plays an important role in the protection of intestinal mucosa (Di and Krantis 2002; Konturek et al. 2004). Moreover, NO has been shown to be involved in neuronal communication, regulation of blood flow and pressure, smooth muscle activity and intestinal motility, modulation of immunity, inflammatory reactions, and the regeneration of axons during injury (Grozdanovic et al. 1994; Belai et al. 1997; Sigge et al. 1998; Bredt 1999; Balemba et al. 2002). An increase in the number of neurons synthesizing NOS was observed under both “native” and experimental intestinally induced inflammation (Boughton-Smith et al. 1993; Miller et al. 1995; Miller and Sandoval 1999). Thus, it is not surprising that NOS expressing IMG neurons became more numerous also in animals with chemically induced colitis (present results). Galanin has been shown to be one the most important substances involved in neuroprotection and in the regeneration and survival of damaged neurons within the central nervous system (Elliott-Hunt et al. 2007). An elevation of the number of GAL-expressing neurons immunoreactivity under various pathological conditions has also been observed by many authors (Ekblad et al. 1998; Ekelund et al. 1999; Liu and Hokfelt 2002; Pidsudko et al. 2008a; Gonkowski et al. 2010). An increased expression of GAL immunoreactivity in intestinal nerve structures may also be connected with the role of GAL in the regulation of pain, where it could
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function as both an analgesic and hyperalgesic mediator. Such suggestion is congruent with the fact that a high increase of GAL expression has been observed within the dorsal root ganglia and spinal cord in pathological pain conditions (Liu and Hokfelt 2002). In the present study, GAL-IR neurons were not observed in IMG of healthy animals but only in pigs with chemically induced colitis, which suggests that upregulation of GAL was induced by colitis per se. In conclusion, the present study indicates that colonprojecting IMG neurons are affected by chemically induced colitis. The number of IMG neurons expressing TH, NPY, SOM, and VIP were downregulated, while the numbers of neurons expressing neuroprotective substances (CB, NOS, and GAL) were distinctly increased. The changes in the chemical coding of IMG neurons observed under pathological condition investigated in this study could reflect adaptative mechanism(s) of the IMG neurons leading to their survival and/or regeneration.
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