Keywords
COVID-19 - choroid plexus - ependymal cell - alcohol - hydrocephalus
Introduction
Sars-Cov-2 (coronavirus disease; COVID-19), as a life-threatening infection, impacts
life globally.[1] Many neurologic and non-neurologic complications have been reported in COVID-19
patients.[2] This pandemic led to a marked reduction in the number of spinal and cranial surgeries.[3]
[4] It is crucial to control this pandemic, but the virus is very contagious and can
survive for at least several days on a variety of materials, and there is no effective
drug against it to date. Without appropriate protection measures, medical professionals
could be exposed to the COVID-19 virus. For that reason, medical professionals and
other people prompted the use of personal protective equipment and skin antisepsis,
such as personal hand cleaning with alcohol. Alcohol can prevent transmission of the
virus, but alcohol exposure may have harmful effects on the central nervous system.
Inhaled alcohol initially bypasses first-pass metabolism and rapidly reaches the arterial
circulation and the brain, and may affect brain structures because the brain is particularly
vulnerable to the damaging effects of alcohol. Hydrocephalus, or the dilatation of
the ventricels, increases the volume of the cerebrospinal fluid (CSF).[5] It may be caused by obstruction of the flow of CSF at any point in its path, irregular
absorption, or, very rarely, excessive production.[5] It is a known disease from ancient times,[6] A comprehensive understanding of pathophysiology is an important issue in medical
practice.[7] Alcohol has complex effects on the brain tissue. Alcohol use may lead to a thinner
cortex and ventricular expansion in healthy individuals as well.[8] However, a detailed study assessing the possible effects of inhaled alcohol on the
development of hydrocephalus in humans is lacking. This study attempts to assess the
effect of alcohol exposure on developing hydrocephalus in rats.
Materials and Methods
Experiment
Inhalation of ethyl alcohol vapor has been performed using the chronic intermittent
exposure method. Twenty-four (∼380 g) male Wistar rats were used. Six rats each were
used as the control group and the sham group. The animals in the sham group was exposed
to water evaporation. The other 12 animals as study groups were divided into two subgroups.
Of these, six (n = 6) were exposed to a low ethyl alcohol dose (500 ppm) and six (n = 6) to a high alcohol dose (2,500 ppm). The animals were exposed to three cycles
a day for 3 weeks in vapor chambers.
Investigation of the Brains
After the experiments, all animals were decapitated under general anesthesia. The
brains of the animals were removed following intracisternal 10% formalin injection.
The ventricle ependymal cells (EC) and choroid plexus (CP) were examined by routine
and glial fibrillary acidic protein (GFAP) immunostaining methods. Brain ventricle
volumes were estimated using the Cavalieri method, and the Evans index was used to
measure the ventriculomegaly. Morphologic changes in the choroidal cells (CC) and
EC were examined according to the study by Yilmaz et al.[9] Degenerated CP ependymal cell numbers were estimated by stereological methods,[10] and the physical dissector method was useds to evaluate the numbers of degenerated
CP EC. Results were compared statistically between groups. All statistical analyses
were performed using IBM SPSS version 22 software package. In all measurements, statistical
differences and significance levels were determined by the Mann–Whitney U test, and the results with a p-value of <0.05 were considered significant.
Findings
Histopathologic Results
[Fig. 1] shows CP epithelial cell number estimation of the stereological method. The stereology
challenge is to clarify the structural inner three-dimensional arrangements based
on the analysis of the structure slices showing only two-dimensional information.[11]
[Fig. 2] shows the CP of the lateral ventricle (LM, H&E, ×10), ciliated extensions of ependymal
(CE), CC, and choroidal artery (CA) in a rat of the control group (LM, H&E, ×20/base).
According to this figure, the CP or EC generate CSF, and the cilia of ciliated cells
beat can generate the functional CSF flow without ventriculomegaly. [Fig. 3] shows the histologic appearances of the partially dilated third ventricle with aqueduct
(blue arrow; LM, H&E, 4/A) and desquamated EC in the ventricle and in aqueduct which caused ventricular
dilatation (LM, H&E, ×10/base) in rats exposed to a low dose of ethyl alcohol. In
this figure, obstruction of the aqueduct and stenosis by minimal ependymal cell desquamation
in the aqueduct can be seen. Ciliopathy likely occurred because the dysfunctional
cilia could not generate CSF flow. [Fig. 4] shows the CP, CC, and EC (LM, GFAP, ×20/base) in an animal of the control group;
ciliated extensions and ependymal cells (A), partial desquamation of a rat exposed to a low alcohol dose (B); cellular angulation, shrinkage, cytoplasmic condensation, basal lamina separation,
and also necrosis are degeneration criteria.[12] These criteria are seen in rate exposed to a high alcohol doese (C; LM, GFAP, ×40/A, B, C). Since ethanol rapidly permeates CP after nasal exposure,
many diverse effects have been observed in rats exposed to a low dose (B) and high
dose (C) of alcohol. [Fig. 5] shows degenerated/deformed choroid plexus (DCP) with deformed/desquamated EC (DEC)
on the desquamated basal membrane in blooded and dilated ventricles are seen (LM,
GFAP, ×10) in the heavy alcohol-exposed rat. In this stage, the disruption of the
CP–brain barrier and CP–blood barrier was likely to occur. The histopathologic view
in [Fig. 5] suggests the disruption of the barriers. The dilated ventricles indirectly show
the CSF accumulation in the ventricles. After blood–CSF barrier disruption, the CSF
exposes many toxic components of blood. [Supplementary Fig. S1] shows degenerated EC just on the desquamated basal lamina (black arrow) and sublaminar edema (LM, H&E, ×10/A); dilated aqueduct with ruptured wall and periaqueductal
edema (LM, GFAP, ×10/B) and DEC with ruptured basal lamina and submembranous hemorrhage
(H/R) are seen in high-dose alcohol-exposed rat (LM, GFAP, ×40/base). One important
finding is the enlarged aqueduct, which led to periaqueductal gray matter fiber rupture,
edema, and capillary hemorrhage. Desquamated ependymal cell mass and cilia dysfunction
are likely causes of ventriculomegaly in the animals exposed to high-dose alcohol.
Probably, the pulsatile component of the CSF flow was lost by occurring ciliopathy.
[Supplemantary Fig S2]; Evans index calculation methos was shown.
Fig. 1 (A, B) The stereological counting method is shown.
Fig. 2 Choroid plexus (CP) of the lateral ventricle (LM, H&E, ×10), ciliated extensions
of the ependymal (CE), choroidal cells (CC), and choroidal artery (CA) are seen (LM,
H&E, ×20/base) in a rat of the control group.
Fig. 3 The histologic appearances of the partially dilated third ventricle with aqueduct
(blue arrow; LM, H&E, 4/A) and desquamated ependymal cells in the ventricle and in aqueduct which
caused ventricular dilatation (LM, H&E, ×10/base) in a rat exposed to a low dose of
alcohol.
Fig. 4 Choroid plexus (CP), choroidal cells (CC), and ependymal cells (EC; LM, GFAP, ×20/base)
in an animal of the control group; (A) ciliated extensions and ependymal cells, (B) partial desquamation of a rat exposed to low doses of alcohol; and (C) cellular angulation, shrinkage, cytoplasmic condensation, basal lamina separation,
and also necrosis are seen in high-dose alcohol-exposed rat (LM, GFAP, ×40/A, B, C).
Fig. 5 Degenerated/deformed choroid plexus (DCP) with deformed/desquamated ependymal cells
on the desquamated basal membrane in blooded and dilated ventricles (LM, GFAP, ×10)
of a high-dose alcohol-exposed rat.
Numerical Results
The mean Evans index was <34% in the control group, >36% in the sham group, >40% in
the group exposed to a low alcohol dose (low-dose alcohol group), and >50% in the
group exposed to a high alcohol dose (high-dose alcohol group). Degenerated epithelial
cell density was found to be 22 ± 5/mm3 in the control group, 56 ± 11/mm3 in the sham group, 175 ± 37/mm3 in the group exposed to a low alcohol dose, and 356 ± 85/mm3 in the group exposed to a high alcohol dose (see [Table 1]).
Table 1
Finding of the study
|
control
|
Sham
|
Low dose alcohol
|
high dose alcohol
|
|
Evans Index
|
<34%
|
>36%
|
>40%
|
>50%
|
|
DECD (/mm3)
|
22 ± 5
|
56 ± 11
|
175 ± 37
|
356 ± 85
|
Abbreviation: DECD, degenerated epithelial cells density.
Note: This table shows our findings which indicate that nasal ethyl alcohol exposure
may cause brain damage by promoting the development of ventriculomegaly in rats.
Discussion
Key Results
It was found that alcohol exposure caused CP and ependymal cell degeneration with
ciliopathy and enlarged lateral ventricles or hydrocephalus.
Corona Virus and the Importance of the Findings of the Present Study
COVID-19 virus infection has a high mortality rate but there is still no definitive
treatment with a drug or vaccine for the Covid-19 virus infection. Various types of
biocidal agents have been used to disinfect surfaces, such as alcohol or benzalkonium
chloride. Vaporized alcohol exposure may have a hazardous effect on the brain. The
findings of the present study revealed a degenerated epithelial cell density of 22 ± 5/mm3 in the control group, 56 ± 11/mm3 in the sham group, 175 ± 37/mm3 in the group exposed to a low alcohol dose, and 356 ± 85/mm3 in group exposed to a high alcohol dose. The Evans index was <34% in the control
group, >36% in the sham group, >40% in the group exposed to a high alcohol dose and
>50% in the group exposed to a hugh alcohol dose We observed that alcohol exposure
caused CP and ependymal cell degeneration with ciliopathy and enlarged lateral ventricles.
According to the finding of the present study, the CP and ependymal cell degeneration
following exposure to alcohol vapor of 100 ppm may not be hazardous but over 5,000 ppm
may be more dangerous for the brain.
Alcohol Abuse and Hydrocephalus
Hickman et al investigated the effect of alcohol consumption on the development of
hydrocephalus.[13] They analyzed 328 patients and found that, overall, 47% of these patients consumed
alcohol to some degree.[13] In this study, it was found that alcohol exposure led to ventriculomegaly and hydrocephalus.
This study is to infer a causal relationship between nasal alcohol exposure and the
development of hydrocephalus. The data of the present study are consistent with a
hypothesis. Alcohol has vasoactive properties,[14] but alcohol also has an unwanted effect on brain structure. The human brain has
several barriers.[15] This barrier is critical to maintaining CNS homeostasis and functions through a tight control of the internal environment free of toxins
and pathogens to provide the proper chemical composition.[16] It was reported that long-term alcohol consumption can damage the blood–brain barrier
integrity.[16] Alcohol abuse may change the permeability of this barrier and may disrupt the blood–CSF
barrier[17] and the blood–brain barrier.
Hydrocephalus after Nasal Alcohol Exposure
This experimental study on 24 male Wistar rats showed the histopathology of CP and
ependymal layer and found bleeding from choroidal blood vessels and damage to EC after
high-dose alcohol inhalation. We arbitrarily preferred the concentration of alcohol
vapor employed in this research. [Fig. 5] shows intraventricular hemorrhage in enlarged lateral ventricles of a rat that was
exposed to a high dose of vaporized alcohol. Having broad knowledge of anatomy is
essential for practicing medicine. CSF is produced by the CP and moved by multiciliated
EC through the ventricular system of the human brain.[13]
[16] CSF is renewed several times a day and is a medium for transportation of nutrients
and signaling molecules and for removing waste products. Excess CSF secretion, obstruction
of the aqueduct, and improper reabsorption of CSF are causes of hydrocephalus. In
[Fig. 2], normal functional EC are seen without ventriculomegaly in a rat of the control
group. In [Fig. 3], minimal ependymal cell desquamation in the aqueduct makes obstruction of the ventricle
and stenosis. [Fig. 3] shows the starting ventriculomegaly which seems to be caused by ependymal cell desquamation.
This study indicates that defects in the ependymal layer, functional disruption, and
ciliopathy secondary alcohol exposure may be the causes of ventriculomegaly.
The effect of alcohol was indirectly assessed by the Evans Index. In humans, the Evans
index is defined as the ratio between the maximal diameter of the frontal horns and
the inner diameter of the skull, and we used this index in the present study. There
are still some concerns about the diagnostic value of the Evans index, but, currently,
it is still an important diagnostic criterion for hydrocephalus. [Supplementary Fig. S2]; Evans index calculation methos was shown. We suggested that CP and ependymal cell
degeneration with ciliopathy may be the cause of ventriculomegaly after alcohol exposure.
This study for the first time shows that nasal ethyl alcohol exposure may cause brain
damage by promoting the development of ventriculomegaly in rats.
Limitation
In this study, we postulated the direct absorption of alcohol from the nasal olfactory
epithelium to the brain. A paired blood alcohol level after exposure to vaporized
ethanol would be crucial to exclude absorption through nasal mucosal via the bloodstream
to the central nervous system. Blood alcohol levels may be different. In the future,
we are planning to measure the blood ethanol level after exposure to vaporized ethanol.
Besides, intracranial pressure (ICP) monitoring can provide additional findings in
this study, but we did not measure ICP. The Evans index is developed for computed
tomography (CT) and magnetic resonance (MR) images normally. Therefore, it might be
interesting to perform an MRI on the rats before the final experiment and fixation
to analyze the ventriculomegaly in vivo and analyze the Evans index. We are planning
such a study by obtaining an MRI of animals before the experiment.
The connection of the study results to COVID-19 is tenuous given the low levels of
exposure with normal disinfectant use. From a toxicologic perspective, the model used
in the study is way beyond pathophysiologic levels. The model uses 2,500 ppm, 15 minutes
a session, and 6 times a day for 3 weeks running. Thus, the physiologic relevance
of our findings may be questionable, but a further cohort with a longer period of
exposure (e.g., 1 month, 2 months) might be helpful to underline the results of this
study.
Another limitation of the study might bes the small sample size. Table 1 shows that
the degenerated epithelial cell density was 22 ± 5/mm3 in the control group and 56 ± 11/mm3 in the sham group. It can be asked why there was an increase in the Evans Index in
the sham group that was exposed only to evaporated water. There was an increase in
the degenerated epithelial cell density in the sham group as well. We think that evaporated
water–related (sham-related) changes have likely occurred. The sham operation also
can be harmful and lead to some changes in animals,[18] as can be seen in this study.
Conclusion
In this study, we investigated the effect of nasal alcohol exposure on the ventricular
system and ventricle size, and it was shown for the first time that nasal exposure
to ethyl alcohol alters the CP EC in the rat brain ventricles. Safety is a critical
element of a drug, besides efficacy. Despite the beneficial role of alcohol in the
control and prevention of COVID-19, there are key concerns regarding the use of alcohol
disinfectants, including the side effects on the human brain and ventricles. All physicians
should note that alcohol including disinfectants should not be used in high concentrations
and long term especially in the Covid-19 pandemic because excessive and long-term
exposure to ethyl alcohol can be a causative factor for ventricular enlargement or
hydrocephalus. There is an urgent need for developing safer and more effective disinfectants
to combat the ongoing Covid-19 pandemic. Plasma-activated water may be efficiently
used as an alternative to conventional alcohol disinfectants to inactivate the Covid-19
virus. More studies are required.
