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Copyright © 2024. Anatomy & Cell Biology
Introduction
The cerebellum carries out multiple important functions
including motor control, motor learning [1, 2] and recent
studies have revealed its roles in aspects of cognitive process-
ing including language, verbal memory [3, 4], executive func-
tioning [5], working memory [6], and emotional processing
[7]. The cerebellum has been noted to express aromatase [8],
and estrogen receptors [9] suggesting that estradiol plays a
role within the cerebellum.
It has been documented that estradiol plays a role in
dendritic growth, spinogenesis, and synaptogenesis in the
cerebellum [10]. Previous studies show that estradiol main-
tains and increases the Purkinje dendritic length, spine
density and arborization [11-13]. Estradiol was demonstrated
to maintain neuronal numbers and morphology in other
regions of the brain such as the inferior olivary nucleus and
the hippocampus [14-16]. Furthermore, exogenous estradiol
administration reduces Purkinje cell death in ethanol – in-
duced cerebellar excitotoxicity [17]. In addition, postmeno-
pausal female receiving estrogen therapy have greater gray
matter volumes in the cerebellum compared to their coun-
terparts not receiving hormonal replacement therapy [18].
Original Article
https://doi.org/10.5115/acb.24.088
eISSN 2093-3673
Corresponding author:
Chaudhry Talha Hannan
Department of Human Anatomy and Medical Physiology, University of
Nairobi, 14 Riverside Drive, Nairobi 30197-00100, Kenya
E-mail: talhahchaudhry99@gmail.com
Histological features of the Purkinje neurons
of the Albino rat (
Rattus norvegicus
) following
letrozole administration
Chaudhry Talha Hannan, Munguti Kilonzo Jeremiah, Pamela Mandela Idenya
Department of Human Anatomy and Medical Physiology, University of Nairobi, Nairobi, Kenya
Abstract: Aromatase inhibitors are increasingly being used as adjuvant therapy for hormone-responsive cancers. These
drugs may reduce the endogenous estrogen production in the cerebellum. Prolonged use has been associated with symptoms
such as ataxia, poorer balance performance and diminished verbal memory, suggesting impaired cerebellar function. Thus,
this study sought to outline the structural basis for the cerebellar deficits observed. Twenty-seven male rats (3 baseline, 15
experimental, 9 control) aged three months were recruited with the intervention group receiving 0.5 mg/kg of letrozole daily
for 50 days by oral gavage while the control group received normal saline. Their cerebella were harvested for histological
processing on days 20, 35, and 50. Photomicrographs were taken and analysed using Fiji ImageJ software. The dendritic spine
densities and Purkinje linear densities were coded and analyzed using IBM SPSS Statistics version 25.0. A P-value of ≤0.05
was considered significant. A temporal decline in the Purkinje linear density as well as pyknosis and cytoplasmic eosinophilia
was noted in the intervention group (P=0.1). Further, the dendritic spine density of the Purkinje neurons in the intervention
group was markedly reduced (P=0.01). The reduction in the linear cell density and the dendritic spine density of the Purkinje
cells following letrozole administration may provide an anatomical basis for the functional cerebellar deficits seen in chronic
aromatase inhibitor use.
Key words: Purkinje cells, Letrozole, Dendrites, Dendritic spines
Received April 3, 2024; Revised August 17, 2024; Accepted August 29, 2024
Anat Cell Biol Chaudhry Talha Hannan, et al
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https://doi.org/10.5115/acb.24.088
This indicates that it may play a neuroprotective role in the
cerebellum.
In recent times, aromatase inhibitors (AI) are increasingly
being used as first line adjuvant treatment for estrogen re-
ceptor positive breast cancer in postmenopausal female [19].
They inhibit the aromatase enzyme hence reduce the avail-
ability of estrogens [20]. Letrozole is a commonly used third
generation non-steroidal AI of high potency and efficacy
[21] that has been established to achieve almost complete
inhibition of aromatase [22] and to penetrate the blood brain
barrier [23]. Due to this, letrozole is expected to inhibit en-
dogenous estradiol production abolishing its neuroprotective
effects on the neurons of the cerebellum. Correspondingly,
use of AIs has been linked to cerebellar defects including
ataxia [24], poorer balance performance [25], cognitive im-
pairment [26, 27], and diminished verbal memory [28, 29].
These adverse effects suggest cerebellar impairment, but
there is paucity of data on the histological changes in the
cerebellar cortex as a consequence of estradiol deprivation
during AI use. Therefore, this study aimed at describing the
histological changes in the cerebellar cortex that may explain
the functional deficits associated with letrozole use.
Materials and Methods
This was a randomized experimental study trial that uti-
lized the rat model. Albino rats were used as the preferred
animal model due to their close physiological and anatomi-
cal similarity to humans, ease of handling [30] and are less
likely to be stressed by human contact [31]. Previous studies
on the effects of estradiol on the cerebellum have used rat
models due to the similar histoarchitecture [32], expression
of aromatase and the expression of estrogen receptors in
the rat cerebellar cortex [11]. Female rats were excluded as
ovarian aromatase is not inhibited by AIs and would act as a
confounder in this study that focuses on the effects of brain
aromatase. No significant sex differences in the expression
and levels of cerebellar aromatase have been demonstrated in
rats and humans [8, 11]. Rats with any demonstrable pathol-
ogy or head injuries were excluded. Absence of pathology
was confirmed by observation and assistance from qualified
animal house attendants. The 2.5 mg Femara (letrozole)
tablets that were utilized were manufactured by Novartis,
Switzerland and were obtained from a local pharmacy store
in Nairobi, Kenya.
Handling and treatment of animals
Twenty-seven male rats of the Rattus norvegicus species
were used in the study. The study was conducted at the De-
partment of Veterinary Anatomy and Physiology animal
house and tissue harvesting and processing of specimen
done at the Department of Human Anatomy, University of
Nairobi Approval to conduct the parent study was sought
from the Biosafety, Animal Use and Ethics Committee, Fac-
ulty of Veterinary Medicine, University of Nairobi (Reference:
FVM BAUEC/2020/249) and conducted according to the
committee’s guidelines. The animals were weighed and then
kept in the study area for one week prior to the start of the
study for acclimatization. They were exposed to a 12-hour
light/dark diurnal cycle and were provided with standard rat
pellets and water ad libitum.
The experimental group were administered with 0.5 mg/
kg of letrozole orally by gavage every day for 50 days. This
dosage was derived from the Food and Drug Administra-
tion -approved dose of 2.5 mg daily by calculation a la Nair
and Jacob [33]. Given that the average duration of letrozole
adjuvant therapy is 5 years [19] and the translation of rat
days to human years is 10 rat days for every human year [34],
an average study intervention period of 50 days was used to
study the long-term effects on the cerebellum. The control
group was administered with normal saline daily by gavage.
On experimental days 20, 35, and 50, rats were randomly se-
lected from the intervention and control group, euthanized,
perfused with formal saline and the cerebella harvested.
Tissue harvesting and processing
The animals were weighed then euthanized by placing
them in lidded containers having cotton wool soaked in
1% halothane. Euthanasia was confirmed by the absence
of heartbeat and ocular reflexes. A midline longitudinal
incision was made on the chest and abdomen and the skin
reflected. The sternum and the ribs were removed to allow
access to the heart for trans-cardiac perfusion. The animals
were then adequately perfused with normal saline to flush
out blood and subsequently followed by 10% formal saline to
fix the body organs including the cerebellum. The rats were
then decapitated and the scalp was cleaned to expose the cra-
nium. The cranium was removed via use of sharp stainless-
steel fine scissors using the optic foramen through the orbit
as an access point. The laminae of the first few vertebrae
were broken using the same stainless-steel fine scissors and
the brain and the attached brainstem were gently extracted
Effects of letrozole on the Purkinje neurons
https://doi.org/10.5115/acb.24.088
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and placed into a container containing 10% formal saline.
The cerebellum was separated from the brainstem by cutting
through the cerebellar peduncles. The cerebellum was divid-
ed into two equal halves with a midsagittal cut. The harvest-
ed tissues were dipped in 10% formalin in specimen bottles
for at least 24 hours before routine processing and staining
for light microscopy. Fifty-four blocks were produced with
one hemicerebellum being stained with hematoxylin & eosin
stain to demonstrate cellular details of the cerebellar cortical
layers, while the other was stained with the modified Patro’s
Golgi stain to elucidate the dendrites of the Purkinje neu-
rons. These blocks were sectioned to produce thirty serial
sections from which every third section was selected to ob-
tain ten standard sections for histomorphometric analyses.
Modified Patro’s Golgi staining technique
The Golgi staining protocol that was utilized has high
specificity for Purkinje cells hence was suitable for this study
[35]. This was to increase the number of Purkinje neurons
stained and minimize glia staining that may produce arti-
facts. A block of cerebellar tissue (≈10×5 mm) was immersed
into a solution containing 5% potassium dichromate, 5%
chloral hydrate, 6% formaldehyde, 2% glutaraldehyde, and 6
drops of dimethylsulphoxide in the dark for 72 hours. 2 ml of
15% sucrose was also added in the chromation step as it has
been shown to improve staining quality and reliability [35].
The block was rinsed with 0.75% silver nitrate solution until
the brick-red precipitate stopped forming on the surface of
the tissue block. The precipitate coat that would have other-
wise prevented impregnation was gently brushed off. It was
thereafter immersed into a 2% aqueous silver nitrate solution
in the dark for another 72 hours. The tissue was subsequent-
ly rinsed with the 2% silver nitrate solution to remove any
precipitate on the tissue surface and dehydrated in ascending
grades of alcohols. It was cleared in toluene for 2 hours and
infiltrated overnight in paraffin wax. The mounted sections
were cut approximately 30 µm thick to avoid cutting through
neurons and their processes. The sections were floated in a
warm water bath to enhance spreading. The sections were
fished from the water bath, onto a gelatinized glass slide that
was prepared using gelatin and chromium potassium sulfate
dodecahydrate. The sections were dewaxed in xylene before
mounting using DPX Mountant (Sigma-Aldrich) and ob-
serving under a light microscope.
Histomorphometric analysis
Out of the 10 serially stained sections, every second se-
rial section was chosen for histomorphometric analysis.
Photomicrographs were taken using a ZeissTM digital photo-
microscope (Carl Zeiss AG) for histomorphometric analysis.
Measures of cell counts from the Purkinje layer from five
random fields, preferentially avoiding areas with sulci and
gyri, on the slides were done and mean values calculated.
The number of Purkinje cell bodies with visibly stained nu-
clei in each photomicrograph were counted. The Purkinje
cell layer length was determined using Fiji ImageJ software
by using the Segmented Line function to draw a line connect-
ing the centers of the Purkinje cell bodies (Fig. 1). Purkinje
linear density (PLD) was calculated using the formula below
[36]:
Five isolated Purkinje neurons per section were identified
and randomly selected based on previously established cri-
teria of effective staining that entails homogenous and com-
plete impregnation throughout the extent of the neuronal
processes, neurons not obscured by neighboring structures
and the terminal dendrites possessing natural terminations
[37]. One dendritic segment was chosen per Purkinje cell
based on an established protocol ensuring each segment was
approximately 10 µm long, unobscured by nearby branches
Purkinje linear density= Number of Purkinje cell bodies
Length of the Purk inje cell layer (in mm)
Fig. 1. Determination of the Purkinje linear density. The number of
Purkinje cell bodies with visibly sta ined nuclei in each photomicrograph
were counted. The Purkinje cell layer length was determined using Fiji
ImageJ software by using the Segmented Line function to draw a line
(yellow line) connecting the centers of the Purkinje cell bodies.
Anat Cell Biol Chaudhry Talha Hannan, et al
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https://doi.org/10.5115/acb.24.088
or structures and that had visible dendritic spines [11, 38].
Photomicrographs at ×1,000 magnification using im-
mersion oil were taken from the Golgi-stained sections. The
dendritic spines whose heads and stems were in focus were
counted. This was determined using Fiji ImageJ software
(National Institutes of Health) (Fig. 2). Dendritic segment
length was measured by drawing a freehand line along the
dendritic segment. The following formula was used [38]:
Statistical analysis and presentation
Histomorphometric data on the PLD and dendritic
spine densities (DSD) was entered into IBM SPSS Statistics
software (version 25.0; IBM Co.) for statistical analysis.
Normality of the data was assessed using the Shapiro–Wilk
test and visual inspection of the histograms, box plots and
normal Q-Q plots generated from the data. The histograms
displayed skewed distributions and, thus, non-parametric
tests were employed. Kruskal Wallis H-tests were employed
to check for statistically significant differences over time in
both the control and experimental group over the study peri-
od. A Dunn Bonferroni post-hoc test was carried out in case
statistically significant differences were noted in the Kruskal
Wallis test. Mann Whitney U-tests were carried out to as-
sess for significant differences in the PLD and DSD between
the control and experimental group on each of the perfusion
days. A P-value of ≤0.05 was considered significant. Data
were presented in tables, photomicrographs and line graphs
were plotted to show the trend observed in the measured
variables.
Results
All the rats used in the study survived until their respec-
tive perfusion timings. The animals gained weight appro-
priately over the study period. At the time of euthanasia, no
discernible gross anatomical differences in cerebella of the
intervention groups were noted when compared to the con-
trol and the baseline groups.
PLD of the control group rose slightly up to day 20 after
which it remained relatively constant over the remainder of
the study period (Fig. 3). The Purkinje cells of the control
group appeared to be uniform in size and some had promi-
nent nucleoli (Fig. 4A, C, E). The photomicrographs revealed
a reduced number of Purkinje cells in the intervention group
with some pyknotic Purkinje cells also being observed (Fig.
4B, D, F). There were no statistically significant differences
between the groups (Table 1). On the other hand, a decrease
in the PLD was noted on administration of letrozole over the
50-day study period. PLD dropped progressively with the
lowest cell density being observed on day 50 (P=0.165).
A marked decrease in DSD of the Purkinje neurons was
observed in the experimental group (Fig. 5). The terminal
dendrites of the Purkinje neurons in the control groups have
a relatively high dendritic spine density (Fig. 6A, C, E). This
was evidenced by a gradual sparsity of dendritic spines on
the terminal dendrite segments with continued letrozole
administration from day 20 onwards (Fig. 6B, D, F). In ad-
dition, the dendritic segments in the experimental group at
day 50 appeared to be more slender (Fig. 6F) as compared to
Dendritic spine density= Number of dendritic spines (n)
Length of the dendritic segment (µm)
AB
Fig. 2. Determination of the dendritic spine density of the Purkinje
neurons. (A) The dendritic spines whose heads and stems were in
focus were counted. The dendritic segment length was measured
by drawing a freehand line along the dendritic segment. (B) The
dendritic spine densities was also confirmed using the corresponding
image skeleton derived using the Skeletonize function of ImageJ.
Day 0 Day 20 Day 35 Day 50
40
35
30
25
20
15
10
5
PLD (cells/mm)
Time (days)
34.04 35.89 35.97 35.82
29.96 30.40
28.34
Purkinje linear density
0
Control
Experimental
Fig. 3. Line graph showing the general trend of the mean Purkinje
linear density over time in the control and experimental groups. PLD,
Purkinje linear density.
Effects of letrozole on the Purkinje neurons
https://doi.org/10.5115/acb.24.088
Anat Cell Biol 5
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A
A
B
B
C
C
D
D
E
E
F
F
Fig. 4. Photomicrographs showing
Purkinje cellular changes following
letrozole administration. Light
microscopic features of the cerebellar
cortex in the control and experimental
groups on days 20, 35, and 50. (A) The
cerebellar cortex of the control group on
day 20. Note the abundance of Purkinje
cells with uniformly staining and
rounded nuclei (H&E, ×400). (B) The
cerebellar cortex of the experimental
group on day 20. Note the pyknotic
Purkinje cells with smaller and darkly
staining nuclei (black arrows). The
number of the Purkinje cells (white
arrows) is lesser than what was seen in
the control group (H&E, ×400). (C)
Cerebellar cortex showing the Purkinje
cells in the control group on day 35.
The Purkinje cells are numerous and are
arranged in a single row. The nuclei are
uniformly basophilic, rounded and have
visible nucleoli (H&E, ×400). (D) The
cerebellar cortex of the experimental
group on day 35. Note the reduction
in number of the Purkinje cells in
comparison to the control group (H&E ,
×400). (E) Cerebellar cortex showing
the Purkinje cells in the control group
on day 50. Note the abundance of the
Purkinje cells (H&E, ×400). (F) The
cerebellar cortex of the experimental
group on day 50. Note the reduced
density of the Purkinje cells (white
arrowheads) as compared to the control
group. Some of the Purk inje neurons are
pyknotic and display basophilic nuclei
and intense cytoplasmic eosinophilia
(black arrowheads) (H&E, ×400). GL,
granular layer; ML, molecular layer.
Table 1. Purkinje l inear density at d ifferent time periods
Day Group PL D (cell s/mm) P-value against
Mean±SD Median Control
Day 0 (baseline) 34.04 ±1.51 33.91
Day 20 Control 35.89±5.11 36.45 0.143
Experimental 29.96±3.68 28.41
Day 35 Control 35.97±1.00 36.46 0.071
Experimental 30.40±4.59 32.10
Day 50 Control 35.82±6.67 33.63 0.071
Experimental 28.34±3.04 27. 5 4
PLD, Purkinje linea r density.
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https://doi.org/10.5115/acb.24.088
the corresponding control group (Fig. 6E). A slight decrease
in DSD of the Purkinje neurons was also noted in the control
group (Fig. 5) but was not statistically significant (P=0.09).
The intervention group displayed statistically significant
temporal differences in the DSD during the study period
(H=12.462; P=0.01). This was noted since the experimental
group at day 50 had a significantly lower DSD when com-
pared to the DSD at day 0 (P=0.01). Upon comparing the
DSD between groups, statistically significant differences
were revealed between the control and the experimental
groups with the latter group having consistently lower values
(all P-values=0.036) (Table 2).
Discussion
This study has shown structural changes in the Purkinje
neurons consistent with the previously noted deficits. Con-
sidering that use of letrozole is on the rise [19], structural ef-
fects of its administration on the cerebellar cortex should not
be taken lightly.
The lower PLD noted on administration of letrozole
was similar to what was described previously by Hill et al.
[39] who used aromatase-knockout mice to mimic a state
of estrogen deficiency. These mice displayed a reduction
in number of the cortical neurons even in the absence of
pathological conditions and external toxic insults. Contrary
to this, some studies have concluded that administration of
an AI alone does not lead to significant effects on the neu-
ronal number in both the adult inferior olivary nucleus and
the neonatal cerebellum [12, 15]. Estradiol has been noted to
have anti-inflammatory activity in the brain that is mediated
via estrogen receptor-beta (ERβ) that is present on Purkinje
cells [40]. It has also been reported to protect Purkinje cells
from glutamate excitotoxicity by promoting Gamma-ami-
nobutyric acid production [41]. A review by Amantea et al.
[42] additionally indicated that it is responsible for vasodila-
tion and increased cerebral blood flow, upregulation of anti-
apoptotic factors such as Bcl-2 and downregulation of pro-
apoptotic factors. The reduction in PLD may be explained
due to letrozole effectively lowering estradiol levels and the
ensuing nullification of its neuroprotective effects. Litera-
ture has also shown that Purkinje neuronal survival may be
linked to antioxidant properties and the phenolic structure
of estradiol that scavenges reactive oxygen species [43].
This reduction in Purkinje cell number may lead to func-
tional deficits as this reduction is commonly seen in various
cerebellar pathologies such as cerebellar ataxia [44]. More-
over, Yang et al. [45] proposed that decreased Purkinje cell
density is a common histological finding in ataxic patients.
Thus, the reduction in PLD possibly explains the ataxia seen
in AI use.
The marked reduction in DSD of the Purkinje cells with
exposure to letrozole seen in our study closely reflects those
seen in estradiol deprivation [15]. In the current study, low-
ering of estradiol levels in the cerebellum may be attributed
to the inhibition of cerebellar aromatase by letrozole. These
findings are in concordance with those of Sakamoto et al.
[11] and Sasahara et al. [12] who reported reduced DSD in
developing murine Purkinje cells secondary to estradiol
deprivation. The role of estradiol in dendritic spinogenesis
has also been elaborated previously in adult rats treated with
exogenous estradiol. These rats exhibited increased DSD of
neurons in other regions of the central nervous system such
as the ventromedial nucleus of the hypothalamus, the basal
forebrain, and the CA1 subfield of the hippocampus [46-48].
Estradiol acts via the nuclear ERβ receptors to upregu-
late levels of brain derived neurotrophic factor (BDNF) [49].
BDNF then increases the levels of tropomyosin receptor ki-
nase B (TrkB) and this pathway leads to increased spinogen-
esis in the Purkinje cells [50, 51] and hippocampal neurons
[52]. Luine and Frankfurt [51] also hypothesized that rapid
changes in the DSD are seen due to the direct actions of es-
tradiol on the synthesis of F-actin, an important component
of the cytoskeleton in the dendritic spine [53]. In addition, it
has been noted that the long-term potentiation leads to phos-
Day 0 Day 20 Day 35 Day 50
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
DSD (spines/ m)
Time (days)
1.67
1.57*
1.13*
1.57*
1.07*
1.42*
0.90*
Dendritic spine density
0
Control
Experimental
Fig. 5. Line graph showing the general trend of the mean Purkinje
dendritic spine density over time in the control and experimental
groups. DSD, dendritic spine densities. *Significant difference
(P-value<0.05).
Effects of letrozole on the Purkinje neurons
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phorylation of cofilin is associated with the stabilization of
the actin cytoskeleton in the dendritic spines. AI have been
noted to impairment of long-term potentiation with subse-
quent dephosphorylation of cofilin and the downregulation
of the dendritic spines on neurons [54].
Decline in the DSD of Purkinje cells has been outlined in
rat models of ataxia and essential tremors [38, 55]. This is not
surprising as a study by Dickstein et al. [56] has shown that
A
A
B
B
C
C
D
D
E
E
F
F
Fig. 6. Photomicrographs showing changes in the dendritic spine density of the Purkinje cells over the study period. (A) Dendrites of the
Purkinje cells in the control group on day 20 displaying the terminal dendrites–possessing dendritic spines–branching off from the smoother
proximal dendrite. The terminal dendrites have a relatively high dendritic spine density (yellow arrows) (modified Patro’s Golgi, ×1,000). (B)
Dendritic structure of the Purkinje cells in the experimental group on day 20 displaying terminal dendrites with decreased dendritic spine
density as compared to the control group (yellow arrow) (modified Patro’s Golgi, ×1,000). (C) Dendrites of the Purkinje cells in the control
group at day 35 displaying terminal dendrites with abundant dendritic spines (yellow arrows) (modified Patro’s Golgi, ×1,000). (D) Purkinje
cells’ dendritic organization in the experimental group at day 35 showing a proximal dendrite giving off terminal dendrites with a lower dendritic
spine density as compared to the controls (yellow arrows) (modified Patro’s Golgi, ×1,000). (E) Dendrites of the Purkinje cells in the control
group at day 50 displaying a proximal dendrite giving off several terminal dendrites. Note the high density of dendritic spines (yellow arrow)
(modified Patro’s Golgi, ×1,000). (F) Dendritic details of the Purkinje cells in the experimental group at day 50. Note the generally thinner
dendrites and the lower dendritic spine density (yellow arrows) (modified Patro’s Golgi, ×1,000). DS, dendritic spines; PD, proximal dendrite;
TD, terminal dendrites.
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reduced DSD has a major impact on neuronal function since
the spines act as a major locus for excitatory neuronal signals.
Therefore, the reduction in the DSD noted in the current study
may provide a structural basis for the functional deficits noted
in chronic AI users due to a reduction in these synapse loci.
The limitations of the current study included the erratic
and unpredictable nature of Golgi method of staining and
the fact that 3D reconstruction of the Purkinje cells was
not carried out leading to underestimation of the dendritic
spines. These were delimited by following a specific Golgi
staining protocol for Purkinje cells to increase the probability
of staining more Purkinje cells and the underestimation was
presumed to be carried through all the dendritic measure-
ments. In addition, the study focused on temporal changes in
DSD upon exposure to the AI rather than absolute numbers
of dendritic spines.
Letrozole administration was associated with the reduc-
tion of the PLD and the DSD of the Purkinje cells. These
structural differences in the cerebellar cortex might provide
the basis for the functional deficits that may be observed in
chronic AI use. Future studies could be carried out to obtain
accurate neuronal densities and DSD using stereological
fractionators and 3D reconstruction of neurons respectively
as these would provide a more accurate quantification of the
Purkinje cells and immunohistochemical studies of the lev-
els of markers of apoptosis, BDNF and TrkB in the adult cer-
ebellum that may indicate the neuromolecular mechanisms
underlying chronic administration of letrozole.
ORCID
Chaudhry Talha Hannan:
https://orcid.org/0000-0001-6508-0521
Munguti Kilonzo Jeremiah:
https://orcid.org/0000-0003-4259-2385
Pamela Mandela Idenya:
https://orcid.org/0000-0003-3940-6983
Author Contributions
Conceptualization: CTH, MKJ, PMI. Data acquisition:
CTH. Data analysis or interpretation: CTH. Drafting of the
manuscript: CTH. Critical revision of the manuscript: CTH,
MKJ, PMI. Approval of the final version of the manuscript:
all authors.
Conflicts of Interest
No potential conflict of interest relevant to this article was
reported.
Funding
None.
Acknowledgements
We are grateful for technical assistance in tissue process-
ing and handling offered by staff at the Histology Laboratory
of the Human Anatomy Department, University of Nairobi.
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Day Group DS D (spi nes/µ m) P-value against
Mean±SD Median Control
Day 0 (baseline) 1.67±0.16 1.67
Day 20 Control 1.57±0.09 1.54 0.036a)
Experimental 1.13±0.12 1.21
Day 35 Control 1.57±0.12 1.51 0.036a)
Experimental 1.07±0.09 1.09
Day 50 Control 1.42±0.08 1.43 0.036a)
Experimental 0.90±0.02 0.90
DSD, dendritic spine densities. a)Stat istically signif icant P-value.
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