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493
Pesq. Vet. Bras. 40(6):493-500, June 2020
Original Article
Animal Morphophysiology
DOI: 10.1590/1678-5150-PVB-6381
ISSN 0100-736X (Print)
ISSN 1678-5150 (Online)
RESUMO.- [
Molossus rufus.] Não há estudos
que caracterizem o sistema nervoso entérico (SNE) destes
à biologia destes indivíduos. A organização e densidade dos
neurônios mientéricos podem variar de acordo com a espécie
animal bem como o segmento do tubo digestório considerado.
O óxido nítrico é um dos principais neurotransmissores
ABSTRACT.- Serenini G.F., Beltrami J.M., Gerônimo E., Favetta P.M., Legnani N.G.E., Otutumi L.K.,
Martins L.A. & Germano R.M. 2020.
Molossus rufus. Pesquisa Veterinária Brasileira 40(6):493-500. Graduate
Program in Animal Science with emphasis in Bioactive Products, Universidade Paranaense,
Praça Mascarenhas de Moraes 4282, Zona III, Umuarama, PR 87502-210, Brazil. E-mail:
prof.ricardogermano@gmail.com
There are no studies that characterize the enteric nervous system (ENS) bats. The
organization and density of myenteric neurons may vary according to the animal species,
as well as the segment of the digestive tube considered. The nitric oxide is one of the key
neurotransmitters present in the myenteric neurons, acting as a mediator in the smooth
muscle relaxation. These neurons are evidenced by immunohistochemistry of nitric oxide
synthase (NOS) or by NADPH-diaphorase histochemistry. In this sense, this study aimed to
characterize the total neuronal population and subpopulation NADPH-d
+
of the myenteric
plexus present in the jejunum of the insectivore species Molossus rufus quantitatively. Five
specimens were collected of M. rufus in a buffer area of the “Reserva Biológica das Perobas” in
segments were collected from the small intestine corresponding to the jejunum intended for
two techniques for neuronal marking, Giemsa and NADPH-diaphorase, and a fragment to
the histological technique of hematoxylin-eosin and Masson’s trichrome. All the procedures
were approved by the “Comitê de Ética no Uso de Animais Unipar” (CEUA - protocol No.
34347/2017) and the “Instituto Chico Mendes de Conservação da Biodiversidade” (ICMBio
- protocol No. 60061-1) The histological sections allowed to highlight the location of the
myenteric plexus between the longitudinal and circular layers of the muscular tunic. The
myenteric plexus had an average of total neuronal population (neurons Giemsa+) of 279.23
neurons/mm2, being the nitrergic neurons (neurons NADPH-d+) represented 20.4% of this
total population, with an average of 58.14 neuron/mm
2
. Therefore, the collected data are
consistent with previous studies in other mammalian species concerning the location of the
myenteric plexus, as well as the neural myenteric proportion NADPH-d
+
compared with the
population of neurons Giemsa+. The gaps in the knowledge of ENS of bats limits comparative
INDEX TERMS: Neurons, myenteric plexus, bats, Molossus rufus, Molossidae, digestive tube, enteric
nervous system, NADPH-diaphorase, morphology.
PVB-6381 MF
molossus rufus1
Grazielli F. Serenini2 , José Matheus Beltrami2, Edson Gerônimo2,
Paula M. Favetta2, Nathalia G.E. Legnani4, Luciana K. Otutumi2,
Lisiane A. Martins3 and Ricardo M. Germano2*
1 Received on October 17, 2019.
Accepted for publication on November 22, 2019.
2 Graduate Program in Animal Science with emphasis in Bioactive
Products, Universidade Paranaense (Unipar), Praça Mascarenhas de Moraes
4282, Zona III, Umuarama, PR 87502-210, Brazil. *Corresponding author:
grazielli.serenini@gmail.com, prof.ricardogermano@gmail.com
3 Veterinary practitioner, Rua da Amizade 488, Recanto Azul, Botucatu,
SP 18603-030, Brazil.
4
Graduate Program in Nutrition, Universidade Paranaense (Unipar), Praça
Mascarenhas de Moraes 4282, Zona III, Umuarama, PR 87502-210, Brazil.
Molossus rufus
morcegos da espécie Molossus rufus].
Serenini G.F., Beltrami J.M., Gerônimo E., Favetta P.M.,
Legnani N.G.E., Otutumi L.K., Martins L.A. & Germano
494
Pesq. Vet. Bras. 40(6):493-500, June 2020
presentes nos neurônios mientéricos, atuando como mediador
no relaxamento do músculo liso gastrointestinal, de modo
que estes neurônios são evidenciados igualmente pela
imunohistoquímica da óxido nítrico-sintase (NOS) ou pela
histoquímica da NADPH-diaforase. Neste sentido, objetivou-
se caracterizar quantitativamente a população neuronal total
e subpopulação NADPH-d+ do plexo mientérico presente
no jejuno da espécie Molossus rufus de hábito alimentar
insetívoro. Foram coletados cinco espécimes de M. rufus em
área de amortecimento da Reserva Biológica das Perobas na
microrregião de Cianorte/PR. Após a eutanásia, em câmara
delgado correspondentes ao jejuno destinados a duas técnicas
para marcação neuronal, Giemsa e NADPH-diaforase e, um
fragmento para a técnica histológica de hematoxilina-eosina
e tricômio de Masson. Todos os procedimentos realizados
foram aprovados pelo Comitê de Ética no Uso de Animais da
Unipar (CEUA - protocolo nº 34347/2017) e pelo Instituto
Chico Mendes de Conservação da Biodiversidade (ICMBio -
protocolo nº 60061-1) Os cortes histológicos possibilitaram
evidenciar a localização do plexo mientérico entre os estratos
longitudinal e circular da túnica muscular. Neurônios Giemsa
+
apresentaram uma média de 279,23 neurônios/mm
2
, já os
neurônios nitrérgicos apresentaram em média 20,4% da
população neuronal mientérica total, sendo evidenciados
58,14 neurônios NADPH-d+/mm2. Portanto, os dados coletados
mostram-se condizentes com estudos anteriores em outras
espécies de mamíferos quanto à localização do plexo mientérico,
bem como, a proporção neuronal mientérica NADPH-d
+
comparada com a população de neurônios Giemsa
+
. As lacunas
existentes quanto ao conhecimento do SNE de morcegos
TERMOS DE INDEXAÇÃO: Neurônios, plexo mientérico, morcegos,
Molossus rufus, molossídeos, tubo digestório, sistema nervoso
entérico, NADPH-diaforase, morfologia.
INTRODUCTION
The bats are animals with acknowledged importance in the
dynamics of ecosystems, given the fact that the wide variety of
food habits (Sazima et al. 1982, Bianconi et al. 2004). Insectivores,
for example, serve as controllers of insect populations (Sipinski
& Reis 1995). Medellín et al. (2000) emphasize that the bats
processes in terms of habitats, being the presence or not of
the bats, an indicator of disturbed areas.
In Brazil, it is reported the occurrence of nine families, 68
genera and 178 species, of these, eight genera and 29 species
are representatives of the Molossidae family, and among the
different species of subfamily Molossinae, there is the Molosus
rufus (Nogueira et al. 2014).
Molossus rufus (E. Geoffroy, 1805), insectivore, being
has as characteristic
number of individuals inhabiting mainly residential buildings
(Peracchi et al. 2011).
The food habits can elucidate, in addition to the characteristics
already known, anatomorphological differences, and it can
ENS is present along the digestive tube since the esophagus
to the anus (Gabella 1990, Furness 2006).
Composed of small sets of nerve cells, enteric ganglia,
(Furness 2012), the submucous and myenteric plexus stand
out, which act in the control of the motility, secretions and
absorption of nutrients (Costa et al. 2000, Schemann & Neunlist
2004, Phillips & Powley 2007), consisting of motor neurons,
interneurons and sensory neurons (Furness & Costa 1980).
The myenteric plexus is located between the longitudinal
and circular layers of the muscular tunic (Furness 2006),
being that the organization and density of neurons may vary
according to the animal species as well as the segment of the
studied digestive tube (Irwin 1931).
Regarding the neurotransmitters expressed in neurons
among them the nitric oxide (NO), produced and released
only when the enzyme nitric oxide synthase (NOS) becomes
active (Furness 2006). It is possible to can sort the neurons
of the myenteric plexus according to the neurotransmitter
associated to it: catecholaminergic or adrenergic, cholinergic
and non-adrenergic-non-cholinergic (NANC) (Paran et al. 2009).
NO is a neurotransmitter that acts as NANC mediator
in the relaxation of the gastrointestinal smooth muscle
(Brookes 1993), being reported as one of the most important
inhibitory neurotransmitters of the intestine (Ekblad et al.
1994, Stebbing 1998, Chen et al. 2002) due to its presence
in different biological reactions (Knowles & Moncada 1994,
Rosselli et al. 1998), as participation in intestinal homeostasis
and elimination of parasites (Halliez & Buret 2015), an
and apoptotic induction (Arantes et al. 2004, Nishikawa et
al. 2007) and as a co-factor in diabetic neuropathy (Stevens
et al. 1995).
Histochemical and immunohistochemical methods
nitregic neurons (Belai et al. 1992, Santer 1994, Saffrey
2004); those who express NOS, in a way that the NADPH-
diaphorase histochemistry (Scherer-Singler et al. 1983) and
the immunohistochemistry of NOS (Fabricius et al. 1996)
show the same distribution of this subpopulation of neurons.
Phillips et al. (2003) highlight that 98% of the total myenteric
population corresponds to the sum of the cholinergic and
nitrergic population. Based on this premise and in the absence
of descriptive studies regarding the ENS of bats, the objective
of this study was to quantify the total population of neurons
of the jejunum of myenteric bats of the species M. rufus as
well as the subpopulation of the neurons NADPH-diaphorase+.
MATERIALS AND METHODS
The collection of the specimens of Molossus rufus was authorized
by “Instituto Chico Mendes de Conservação da Biodiversidade”
(ICMBio - Protocol No. 60061-1) and the procedures were approved
by the “Comitê de Ética no Uso de Animais Unipar” (CEUA - protocol
number 34347/2017).
Five specimens of bats Molossus rufus
were collected, in residence of the rural area located in the municipality
of São Tomé, in the micro region of Cianorte, Paraná State, Brazil.
The residence in question housed a large colony of molossidae,
in view of the proximity with the urban area and riparian forest.
495
Pesq. Vet. Bras. 40(6):493-500, June 2020
Molossus rufus
Biological Reservation of Perobas (Brasil 2012) (Fig.1), characterizing
an ecological corridor for different species. The exact location of the
collection area was designated via GPS (Global Positioning System),
being 2351’38.99’’S and 52
Two mist nets positioned in places of the animals’ leaving, before
sunset were used. The bats started their leaving around 06:00 p.m.,
so that the number of required specimens were obtained along a
time of collection, after this period, the nets were withdrawn.
The animals were collected randomly, regardless of sex, being
captured was performed according to Vizzoto & Taddei (1973),
Gregorin & Taddei (2002) and Ramos et al. (2013).
In order to minimize possible causative factors of stress, the
animals were housed in cotton individual bags so that they could be
sent to the Laboratory for Experimental Morphology of the Graduate
Program in Animal Science with emphasis in Bioactive Products of
“Universidade Paranaense”, Umuarama, Paraná.
The euthanasia of each animal was carried out in camera
laparotomy was performed by the middle line of the anal region
until the sternum. The collection of small intestines, corresponding
to the jejunal segment was divided into two fragments of equal size,
so that each one was subjected to two techniques for marking of
neurons. A third fragment of jejunum, half a centimeter, was intended
to routine histological processing, stained by hematoxylin-eosin (HE)
+
Fragments of the jejunum
of each animal were intended to evidence the total population of
myenteric neurons Giemsa
+
. The segments were washed with saline
in this solution until obtaining the membrane formulations. To keep
removed, and this was sectioned along the longitudinal axis at the
level of the insertion of the mesentery. Next, each fragment was
micro dissected to the stereomicroscope with transillumination,
removing the mucosal tunic as well as the submucous mesh. The
muscle and serous tunics were preserved, which constituted the
membrane formulations.
Each membrane was immersed in a solution containing Giemsa
dye methylene blue in phosphate buffer of Sorense (pH 6.9) for
18 hours at ambient temperature. Then, they were dehydrated in
sequence of alcohols (95% and Absolute I for one minute each,
between slide and glass plate with synthetic resin.
+
Fragments of the jejunum collected were subjected to histochemical
technique of Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase
(NADPH-d). For this, the segments of jejunum were washed with
phosphate buffer solution (PBS) (pH 7.4). After that, the edges were
the sequence, the fragments were immersed in paraformaldehyde
4% for 30 minutes and then washed in PBS containing Triton X-100
0.3% for 10 minutes.
After that, the fragments of the jejunum were again washed,
for three more times (10 minutes each), in PBS and incubated for
90 minutes in the reaction medium containing 50mg of Nitro Blue
Tetrazolium
Tris-HCl buffer (0.1M, pH 7.6). After the permeabilization, segments
each), and at the end of this period, the suture wires were removed
on one end and the fragments were immersed in a solution of 4%
storage.
Later, the fragments of the jejunum were sectioned along the
longitudinal axis at the level of the insertion of the mesentery and
from these, membrane formulations were obtained, following the
same procedures already described for the microdissection technique
of Giemsa staining.
The obtained membrane preparations followed for dehydration in
ascending sequence of alcohols (80%, 90%, Absolute I and Absolute II)
followed of diaphanization in xylol (I and II). After concluding such
procedures, the membrane formulations were placed between slide
and glass plate with synthetic resin.
The delimitation of the mesenteric,
intermediate and anti-mesenteric areas of the jejunum was done in
accordance with Sant’Ana et al. (1997), as a way of guidance for the
capture of images, in order to sample the three areas equally. The
obtained material was visualized under light microscope (Nikon
Eclipse E200), with a 40x objective lens, and a system of image
analysis coupled to high resolution camera (Moticam 5.0 megapixels),
being transferred to the computer.
membrane being contemplated the mesenteric, intermediate and
anti-mesenteric areas of the jejunum, intended for the quantitative
analysis of the myenteric neurons stained by Giemsa technique and
by histochemistry NADPH-d+. Halves neurons were considered in
Fig.1 Location of the Biological Reservation of Petrobras, coverage
area and buffer zone. The municipality of São Tomé (X), in the
micro region of Cianorte, represents the site of the collection
area. Adapted from: Brasil 2012.
496
Pesq. Vet. Bras. 40(6):493-500, June 2020
The obtained data were subjected to
descriptive analysis, using the program BioEstat 5.0 (Ayres et al.
2007). The number of total myenteric neurons (Giemsa+) and the
number of nitrergic myenteric ones (NADPH-d+), per mm2, were
obtained for each animal, and the mean, standard deviation and
RESULTS
The analysis of the histological sections revealed the location
of the myenteric plexus for Molossus rufus between the
longitudinal and circular layers of muscular tunic and the
Observed to the light microscope, neurons of the myenteric
plexus of M. rufus were organized into ganglia mostly composed
of several cell bodies, but isolated neurons were also found
area sampled for each fragment of jejunum was 3.66mm
2
the neuronal density per mm2 specimen studied.
In Table 1 the following are expressed: the means ± standard
of total myenteric neurons (Giemsa
+
) and subpopulation
NADPH-d+ of the jejunum of M. rufus estimated per mm2 of
each specimen studied.
For the membrane formulations stained by Giemsa technique
an average was evidenced of 279.23±32.42 neurons/mm
2
and, for the histochemistry NADPH-diaphorase, average of
58.14±17.48 neurons/mm2 (Table 1).
DISCUSSION
neurons of the jejunum of a species of insectivore bat, and there
not being data in the literature regarding the characteristics
of Enteric Nervous System (ENS) for bats in general, different
studies with animal models were used, not only mammals,
presuming the phylogeny occupied by different groups.
The location of the myenteric plexus of Molossus rufus
is consistent with the described by Furness (2006), which
highlights the importance of preserving the muscular tunic
in the process of obtaining the membrane formulations, once
that the myenteric plexus is located between the longitudinal
and circular layers of muscular tunic.
Characteristics of the ganglionic arrangement of myenteric
plexus are described in several studies (Sant’Ana et al. 1997,
Germano et al. 2000, Stabille et al. 2000, Yang et al. 2013)
that mention differences in this arrangement depending on
the intestinal segment and the species studied (Irwin 1931,
Furness & Costa 1980, Gabella 1990, Furness 2006) as well
as in different food habits (Stabille et al. 2002, Münnich et
al. 2008, Previato do Amaral et al. 2017).
in M. rufus
al. (2000).
The specimens collected in spite of presenting different
genders, four males and one female, it has been observed
that for M. rufus the ganglionic organization of myenteric
neurons does not differ among the individuals, the neurons
were grouped into ganglia, having the same irregular formats
that interconnect the ganglia.
Assuming the phylogenetic evolution of the ENS, consider
of collection of the studied intestinal fragments and body
morphofunctional and quantitative alterations of the myenteric
plexus in animals with different ages, between the different
intestinal regions, as well as the organ region sampled, are
Fig.2 Population of total myenteric neurons (Giemsa+) and subpopulation of myenteric nitrergic neurons (NADPH-d+) evidenced by mm2
per animal.
Molossus rufus2.
Technique Mean S.D. C.V.%
Giemsa 279.23 ±32.42 11.61
NADPH-d 58.14 ±17.48 30.08
497
Pesq. Vet. Bras. 40(6):493-500, June 2020
Molossus rufus
essential to the understanding of the above considerations. It is
therefore conjectured that increased or decreased expression
of NADPH-d+ neurons correlates not only with metabolic
status, but also with age, compensation for neuronal loss,
and region of intestinal specimen collection.
Furlan et al. (2002) in morphoquantitative study with
were found grouped in ganglia, being rare, isolated neurons.
Germano et al. (2000) observed ganglia of sparse distribution,
containing two or more neurons, and also isolated neurons
in Cyprinus carpio, highlighting the location of the myenteric
plexus between the longitudinal and circular layers of
muscular tunic.
Previato do Amaral et al. (2017) in exploratory and
descriptive study of the myenteric plexus of broilers (Gallus
gallus domesticus) for NADPH-d+ neurons in the duodenum,
evidenced nitrergic neurons in both, ganglion arrangements
the ganglia.
The data found for M. rufus bats in this study are similar
to those described by the authors for the respective species
studied, Rattus norvegicus, C. carpio and G. gallus domesticus
authors, as well as those from this study, converge to aspects
of the phylogenetic evolution already described for the enteric
nervous system (Furlan, 2000). Different studies reinforce
(Germano et al. 2000) and from chickens this conformation
is inverted, with myenteric neurons predominantly organized
in ganglia (Gabella & Halasy 1987).
Regarding the neuronal disclosure of proximal colon of
adult rats, Furlan et al. (2002) showed an average of 5122
neurons Giemsa
+
in 17.68mm
2
, these being corresponding to
an average of 289.70 neurons/mm2. Germano et al. (2000),
neurons Giemsa
+
in 6.92mm
2
, equivalent to 294.79 myenteric
neurons Giemsa
+
/mm
2
. Compared to the results of this study,
the neuronal population Giemsa
+
among the different species
are similar (Table 1).
Ferezin et al. (2017) reported the count of approximately
45 neurons Giemsa+ in 0.249mm2, corresponding to 180.72
neurons/mm2
, and 12 neurons NADPH-d+ in 0.249mm2,
equivalent to 48.19 neurons/mm2 for the colon of rats.
Previato do Amaral et al. (2017) found a mean ± standard
deviation of 24.38±4.97 neurons NADPH-d
+
/mm
2
in the
duodenum of broilers, values which were lower than the
values found in the present study, 58.14±17.48 neurons
NADPH-d+/mm2.
Yang et al. (2013) found 58.88±3.16 neurons NADPH-d+/
mm2 in the jejunum of broilers aged 15 days, and 45.92±17.51
neurons NADPH-d
+
/mm
2
for broilers aged 40 days. Compared
to the values of nitrergic neurons found in M. rufus, the results
were similar although differences are expected among the
species.
The mean of inhibitory neurons, as evidenced in this
study, corresponds to 20.4% of the population of myenteric
neurons evidenced by Giemsa technique, it is highlighted
that the results fall within the expected parameters, once
that they resemble those of Ekblad et al. (1994), who found
21% of neurons containing NOS in the small intestine of rats.
Other studies have found a percentage next to those, 23% for
guinea-pigs (Furness 2006), 29% in rats (Qu et al. 2008) and
A smaller number of marked inhibitory neurons (nitrergic)
does not necessarily indicate a lower number of nitrergic
neurons, because the enzyme present in the reaction of
the neuronal nitrergic marking in the NADPH-d technique,
shows only those neurons with NOS activity at the time of
the reaction (Scherer-Singler et al. 1983).
Considering that for the total neuronal myenteric population,
the studied individuals, differing only concerning the number
of nitrergic neurons in activity at the time of collection.
Regarding the studies on the bats’ digestive tube, they stand
out for their histology (Makanya et al. 2001, Gadelha-Alves et al.
2008, Strobel et al. 2015, Zhang et al 2015) and microbiology
(Ingala et al. 2018, Sens Junior et al. 2018). However, Barry-
Jr (1976) already emphasized the little research about the
different segments of this system, including the intestine to
wild mammals, a real fact still today, once that the studies on
ENS are widely disseminated to rodents (Karaosmanoglu et
al. 1996, Tan et al. 2008, Luesma et al. 2013, Grundmann et
al. 2015, Kulkarni et al. 2017) and more recently to primates
(Noorian et al. 2011), but in terms of other groups of wild
mammals, not employed in experimental models, the absence
of data is notorious.
Gadelha-Alves et al. (2008), emphasizes the importance of
the study of the bats’ digestive system, aiming to understand
the factors associated with it, such as environmental changes
originated from anthropic action in their habitats.
Zhang et al. (2015), upon comparing the surface of the
mucosa of the small intestine of Tadarida brasiliensis, also an
insectivore bat, and the species Mus musculus discussed the
high digestive capacity of bats in association with the higher
density of villi and enterocytes, corresponding to a higher
paracellular permeability per cm2 in the bats. The authors also
emphasize to the digestive capacity and its relation with the
complex of high capacity, despite their small body mass.
In this sense it is worth mentioning that the myenteric
neurons coordinate the intestinal movements, interfering
not only in the intestinal transit, but also in the absorption
capacity (Costa et al. 2000, Schemann & Neunlist 2004,
Phillips & Powley 2007).
Considering the time of the capture of animals for this
study before the start of the foraging, a lower inhibitory
neuronal expression can be considered. Thus, the density of
nitrergic inhibitory myenteric neurons is not related only to
morphological adaptations and the diet of the animal, but also
with its metabolic state at the time of marking those neurons,
i.e., whether the neurons that express NOS are active or not
(Scherer-Singler et al. 1983).
Gabella (1990) emphasizes that the extension of the
importance of the functions of the same for the survival
of the animal especially in nature, in view the high level of
adaptability to variation in food contents and feeding time,
require a well-developed nervous control.
However, the absence of an effective blood-neuron barrier
(Furness 2006) and the high paracellular absorption capacity
of the bats (Fasulo et al. 2013) leave these animals very
498
Pesq. Vet. Bras. 40(6):493-500, June 2020
susceptible to exposure to toxins present in environments
subject to change by human occupation, and by chance, in
their food, which could compromise the neuronal population.
It is therefore highlighted the importance of this exploratory
and quantitative study of myenteric neuronal population of
bats, corroborating with a better understanding of these
individuals in their entirety.
CONCLUSIONS
The myenteric plexus in bats of the species Molossus
rufus is located between the longitudinal and circular layers
of muscular tunic, while neurons organized into ganglia
Variations in the total population of myenteric neurons is
expected among the different species as well as in different
segments of the digestive tube. The mean of the subpopulation
of active nitrergic neurons in the studied species is consistent
with that described for different species of animals of different
classes, assuming a variation from 20 to 35%.
neurons and the nitrergic subpopulation contributes to
know the morphology and function of the enteric nervous
system (ENS) in bats, since these are distinguished from
in understanding the intestinal anatomy peculiar to these
individuals, described as proportionately small, however
with great digestive capacity.
These results can contribute to future research aimed
at a better understanding of these animals in the context of
unique health.
- The authors are grateful to “Universidade Paranaense”
(Unipar) that through “Diretoria Executiva de Gestão da Pesquisa e Pós-
Graduação” (DEGPP) supported the development of this research and to
“Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES),
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