Histological assessment of cerebellar granule cell layer in postmortem brain; A useful marker of tissue integrity?

Abstract

Tissue quality control measures are routinely performed in brain banks with the assessment of brain pH being the most common measure. In some brain banks the assessment of the RNA integrity number is also performed, although this requires access to specialised equipment and is more expensive. The aim of this study is to determine if there is a correlation between the visual assessment of cerebellar granule cell integrity and brain pH or RIN. One hundred and five consecutive cases from the NSW Tissue Resource Centre, Sydney, Australia were accessed. The cerebrum was hemisected and one hemisphere sliced parasagittally at approximately 1-2 cm intervals and frozen. The other hemisphere was fixed in 15% buffered formalin for 2-3 weeks. The contralateral cerebellar hemisphere was preserved in the same manner as the cerebral hemisphere. Samples of fixed tissue were embedded in paraffin, 7 μm sections cut and stained routinely with hematoxylin and eosin. The granular cell layer (GCL) was assessed microscopically to determine the degree of autolytic degradation. Degradation was graded as nil, mild, moderate or severe. Brain tissue pH and RIN were measured using standardised protocols. This study showed that both brain pH and RIN significantly correlated with the severity of the degradation of the cerebellar granule cell layer. This additional screening tool can be performed during routine histological review of the cerebellar tissue to assess the suitability for further investigation of tissue quality.

Full text

Histological assessment of cerebellar granule cell layer in
postmortem brain; a useful marker of tissue integrity?
Donna Sheedy,
Discipline of Pathology, The University of Sydney, Sydney, NSW 2006, Australia
Antony Harding,
Discipline of Pathology, The University of Sydney, Sydney, NSW 2006, Australia
Meichien Say,
Discipline of Pathology, The University of Sydney, Sydney, NSW 2006, Australia
Julia Stevens, and
Discipline of Pathology, The University of Sydney, Sydney, NSW 2006, Australia. Schizophrenia
Research Institute, Sydney, Australia
Jillian J. Kril
Discipline of Pathology, The University of Sydney, Sydney, NSW 2006, Australia. Discipline of
Medicine, Sydney Medical School, The University of Sydney, Sydney, Australia
Jillian J. Kril: jillian.kril@sydney.edu.au
Abstract
Tissue quality control measures are routinely performed in brain banks with the assessment of
brain pH being the most common measure. In some brain banks the assessment of the RNA
integrity number is also performed, although this requires access to specialised equipment and is
more expensive. The aim of this study is to determine if there is a correlation between the visual
assessment of cerebellar granule cell integrity and brain pH or RIN. One hundred and five
consecutive cases from the NSW Tissue Resource Centre, Sydney, Australia were accessed. The
cerebrum was hemisected and one hemisphere sliced parasagittally at approximately 1–2 cm
intervals and frozen. The other hemisphere was fixed in 15% buffered formalin for 2–3 weeks.
The contralateral cerebellar hemisphere was preserved in the same manner as the cerebral
hemisphere. Samples of fixed tissue were embedded in paraffin, 7 μm sections cut and stained
routinely with hematoxylin and eosin. The granular cell layer (GCL) was assessed microscopically
to determine the degree of autolytic degradation. Degradation was graded as nil, mild, moderate or
severe. Brain tissue pH and RIN were measured using standardised protocols. This study showed
that both brain pH and RIN significantly correlated with the severity of the degradation of the
cerebellar granule cell layer. This additional screening tool can be performed during routine
histological review of the cerebellar tissue to assess the suitability for further investigation of
tissue quality.
Keywords
Postmortem; Brain pH; RIN
© Springer Science+Business Media B.V. 2011
Correspondence to: Jillian J. Kril, jillian.kril@sydney.edu.au.
NIH Public Access
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Introduction
Brain banks provide important support and enhance research into neurological and
psychiatric disorders. Paramount to the management of these facilities is the adherence to
standardised protocols for brain collection, dissection, classification and storage. Unlike
animal studies premortem events are a major contributor to the variability in tissue quality.
Recognised premortem factors such as co-existing pathology (Sheedy et al. 2008), hypoxia
and coma effect the histological and molecular structure of the brain (Harrison et al. 1995).
Exclusion criteria for donation can limit the effect of premortem factors to a large degree,
however it may sometimes be difficult to determine this information at time of death.
In any bio-bank the operational costs are considerable to ensure that the collection,
classification, integrity and long-term viability of each case are of the highest quality. The
measurement of brain pH is a widely recognised marker of brain tissue quality that is
utilised in most brain banks to assist in the allocation of research cohorts (Kingsbury et al.
1995; Monoranu et al. 2009). The positive correlation between low brain pH and degraded
RNA has been reported previously and validates the use of pH as a marker of tissue integrity
(Kingsbury et al. 1995; Monoranu et al. 2009; Schroeder et al. 2006; Webster 2006).
The RNA quality marker, as indicated by the RNA integrity number (Jolles et al. 1992), for
molecular studies is recognised as more reliable predictor of RNA integrity than the
previously employed ratio of 28S:18S ribosomal RNA (Schroeder et al. 2006; Weis et al.
2007). However, the measurement of mRNA, DNA and protein degradation requires access
to specialised instruments and these can be expensive or unavailable to some banks.
A number of previous studies have shown that neurons of the cerebellar granule cell layer
are sensitive to postmortem interval, agonal state and to the conditions of postmortem tissue
storage (Albrechtsen 1977a, b; Ikuta et al. 1963; Ogata et al. 1986). A previous report of a
pilot study from NSW Tissue Resource Centre (NSW TRC) showed a correlation between
the severity of granule cell layer autolysis and a lower mean brain pH (Sheedy et al. 2008).
The NSW TRC has a primary focus on the collection of neurological disorders associated
with alcohol abuse; psychiatric disorders such as schizophrenia and bipolar disorder;
neurodegenerative diseases such as motor neuron disease, and multiple sclerosis, as well as
healthy controls. As the current NSW TRC quality control protocol incorporates the
measurement of brain pH and RIN this study investigated the relationship between the visual
assessment of the granule cell layer of the cerebellum and brain pH and RIN. Histological
assessment of granule cells may provide an additional quality screening assessment tool to
incorporate in routine diagnostic screening procedures.
Methods and materials
One hundred and five consecutive cases collected by the NSW TRC over a 20 month period
were assessed. The NSW TRC has approval from the Human Research Ethics Committee of
Sydney South West Area Health Service (X07-0074 and X08-0065) and The University of
Sydney. Collection and dissection protocols have been previously reported (Sheedy et al.
2008). In brief, cases are donated through either a prospective donor program or a forensic
institution. The NSW TRC protocols have exclusion criteria for donation to reduce the
potential of tissue degradation. These include artificial ventilation with 24 h of death, coma,
protracted agonal period, stroke, head trauma and postmortem interval greater than 72 h. At
postmortem, the cerebellum and brainstem are removed from the cerebrum and hemisected.
One cerebellar hemisphere is sliced parasagittal at approximately 1–2 cm intervals and
frozen. The other hemisphere is fixed in 15% buffered formalin for 2–3 weeks. Routine
samples of fixed tissue, including the lateral cerebellar hemisphere, were embedded in
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paraffin, 7 μm sections cut and stained with hematoxylin and eosin for neuropathological
assessment. Diagnosis was performed using standard protocols.
Brain tissue pH
A small segment (approximately 3 g) of the lateral cerebellar hemisphere was taken at the
time of the autopsy and frozen at −80°C. Brain tissue pH was measured using one gram of
tissue homogenized (IKA T10 basic Homogeniser; Crown Scientific) in 10 volumes of
distilled water at neutral pH (Kingsbury et al. 1995). Measurements were made in triplicate
using the Mettler Toledo MP220 temperature compensating pH meter and the mean value
calculated. Two calibration points were used, pH 4 and pH 7.
RNA integrity
Assessment of the RIN, for assigning integrity values to RNA measurements, was
performed as employed in other banks (Miller et al. 2004; Schroeder et al. 2006; Stan et al.
2006; Webster 2006). In brief, frozen cerebellum samples (30 mg) were homogenised using
the IKA T10 basic Homogeniser (IKA®-Werke GmbH & Co, Cat. #3420000, Germany).
Total RNA extraction was carried out using the Purelink RNA Mini Kit (Invitrogen by Life
Technologies, Cat. #12183018A, USA), according to the manufacturer’s instructions.
Following extraction, RNA was eluted into 60 μl of supplied RNAse/DNAse free water.
Aliquots of 2 μl were sampled for measurement of RNA concentration using the Nanodrop
ND-1000 Spectrophotometer (Nanodrop Technologies, USA). For quality assessment, 5 μl
aliquots samples were electrophoresed according to the Agilent Bioanalyser 2100 (Agilent
Technologies, USA) manufacturer’s protocol (provided in the RNA Nano 6000 kit) for this
analysis. The 2100 Bioanalyzer Expert software then automatically determines the RIN, a
quantitation estimate and the 28S/18S ribosomal ratio. The RIN reflected the presence or
absence of degradation products; where higher RNA degradation was assigned a lower RIN
value.
Histological assessment of autolytic change in cerebellar granule cell layer
The granular cell layer of the cerebellum was assessed microscopically to determine the
degree of autolytic degradation by one of the authors (AH) blinded to case information.
Degradation was graded as nil, mild, moderate or severe as described by Ogata et al.
(Albrechtsen 1977a, b; Ogata et al. 1986). Briefly, these categories are defined as; nil when
no morphological abnormality was detected and granule cells nuclei were clearly visible;
mild when there is a slight decrease in the number of the nuclei but remaining nuclei were
clearly identified; moderate when there is a diffuse decrease in the number of nuclei but
some individual nuclei are still clearly seen and severe when few or no nuclei are clearly
seen and there is vacuolation of the granule cell layer (Fig. 1).
Clinical indices
Postmortem clinical characterisation is performed in accordance with Diagnostic and
Statistical Manual of Mental Disorders (DSM-IV) diagnosis. Cases that are unable to have a
diagnosis confirmed are excluded from the main research cohorts. The mode of death or
agonal duration was determined by assessment of the rapidity of death and related
circumstances (Tomita et al. 2004). These were classified as:
rapid
- a terminal phase of less
than 1 h;
intermediate
- a terminal phase of between one and 24 h and
long term
- longer than
1 day. Postmortem interval (death until freezing of tissue) was calculated in hours for all
cases.
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Statistical analysis
Group effects were tested using analysis of variance (ANOVA) and Fisher’s protected least
squares difference (PLSD) was used for
post hoc
analysis where appropriate. Standard linear
regression was used to assess the relationship between postmortem interval; mode of death;
granule cell layer classification and the tissue quality measures of brain pH and RIN (a
P
value of <0.05 was used for significance). Analysis was performed using JMP statistical
software package (SAS Institute, Cary, NC).
Results
The cases were allocated to the following diagnostic groups: alcohol abuse disorders,
controls, motor neuron disease (MND), multiple sclerosis (Mexal et al. 2006), psychiatric
disorders, and “other” (cases with an unconfirmed clinical diagnosis). Psychiatric disorders
group includes cases of DSM-IV diagnosis of schizophrenia, bipolar and major depression.
Alcohol abuse disorders group includes cases DSM-IV diagnosis of harmful use, substance
abuse (alcohol) and substance dependence (alcohol). Of the 105 cases, 26 were donated
through the prospective donor program and the remainder from the forensic donation
process. Case DSM-IV classification was not significantly correlated with either brain pH,
RIN or GCL. Post mortem interval (PMI) ranged from 3 to 72.5 h.
There was a positive correlation between brain pH and RIN (F1,101 = 69.11,
P
< 0.0001). No
significant correlation was seen with PMI and RIN (
P
= 0.383). A correlation with PMI and
brain pH (y = 6.35 + 0.0053, r2 = 0.10
, P
= 0.0013) within the whole group was observed.
The longer the PMI the brain pH increased. Further post hoc analysis of the diagnostic
groups showed this observation was more prominent in the alcohol abuse disorders and MS
groups though not highly significant,
P
= 0.05 and
P
= 0.07 respectively (Table 1).
Granule cell layer
Grading of the cerebellar granule cell layer determined there were 91 normal cases; 6 with
mild changes; 5 with moderate change and only 3 with severe autolysis. The mean values
for the quality markers brain pH and RIN for each category are shown in Table 2.
Brain pH
As shown in Fig. 2a there was a reduction in the mean of brain pH in relation to the severity
of degradation of the granule cell layer. This is highlighted in the comparison of the mean
values for the normal GCL grade 6.60 (±0.23) to the severe grade 5.77 (±0.15). Oneway
ANOVA showed significance for brain pH (F3,103 = 24.13,
P
< 0.0001). Post-hoc analyses
revealed the severity of the degradation of the GCL showed significance for brain pH when
compared to the normal GCL; mild
P
= 0.0004, moderate and severe both
P
< 0.0001. The
comparison between mild grade of autolysis to severe was significant
P
= 0.005 as well.
RIN
The mean RIN values as shown in Fig. 2b also decreases with the severity of the GCL
degradation. The mean RIN of the normal GCL grade was 7.30 (±0.90) as compared with
the severe grade, mean 1.6 (±2.77). Oneway ANOVA for the RIN (F3,103 = 37.44,
P
<
0.0001) displayed significance. Further post-hoc analyses found the RIN was affected by the
severity of degradation. The comparison of normal, mild and moderate groups to the severe
group showed to be highly significant (
P
< 0.0001). There was an effect between the normal
and moderate groups
P
= 0.0027. No significant difference was seen for normal to mild or
mild to moderate.
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Mode of death
The mode of death in the total cohort comprised of the ratio; 45% rapid, 30% intermediate
and 25% long term. Due to the characteristics of the disease, the cases with MND and MS
had a larger proportion (62%) with a long agonal phase. Of the three severe GCL cases, only
one had a long-term agonal phase, the other two had an intermediate phase. The effect of
mode of death on GCL was not significant; Chi Sq
P
> 0.367.
Discussion
Brain banks endeavour to provide material of high quality for a range of research
techniques. The adherence to standardised protocols assists in meeting this goal. Allocation
of cases to study cohorts is also dependent on brain tissue quality markers; therefore having
an additional screening tool assists in the assessment of case suitability.
The normal density of the cerebellar granule cells in humans is approximately 107 per mm3
and during autolysis, cell lysis proceeds selectively leading to a lower pH as increased
lysosomal enzyme release leads to increase loss of remaining cells (Albrechtsen 1977a, b;
Averback 1980). Changes seen in the cerebellar granule cell layer without glial reaction
have been shown to be a postmortem event (Albrechtsen 1977a, b). In previous pathological
studies of human postmortem cerebellar cortex, the activity of the proteolytic enzyme,
naphthylamidase (LNAse), is consistently shown when the pH was at an optimum of 5.8
(Albrechtsen 1977a, b). This mainly occurred in the granule cell layer of the cerebellar
cortex. Our study shows a similar finding in the cases with severe degrading of the GCL
where the mean brain pH of was 5.77.
Brain tissue pH is an important factor in the interpretation of molecular data. Gene
expression studies have shown strong correlations to brain pH (Li et al. 2004; Mexal et al.
2006; Tomita et al. 2004; Weickert et al. 2010). Expression patterns exhibited by samples
with lower pH tend to involve energy metabolism and proteolytic activities. This increases
the genes encoding stress related response proteins and transcription factors (Li et al. 2004).
The ability to provide study cohorts that have been reviewed for quality integrity is essential
to postmortem research studies and the interpretation of their results. Even though there
were only a small number of cases with moderate or severe changes to the granule cell layer
the robust correlations with pH and RIN suggest that the visual assessment of GCL may be a
useful and inexpensive screening tool. Review of the histopathological changes of the
cerebellar granular cell layer can be performed during the routine neuropathology
examination, without adding time or complexity to the screen process. Indeed, the visual
assessment of the granule cell layer may reduce the necessity of performing expensive RNA
integrity analyses in those cases that have severe cerebellar granule cell layer autolysis.
Combined with the measurement of brain pH, the assessment of the cerebellar granule cell
layer may assist banks that do not have facilities to perform the extraction for RNA
integrity.
Acknowledgments
The NSW TRC is supported by the University of Sydney, National Health and Medical Research Council of
Australia
(605210)
, Schizophrenia Research Institute, and the National Institute of Alcohol Abuse and Alcoholism
(
R24AA012725
).
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Fig. 1.
Grading scheme used to assess the increasing severity of autolysis of the cerebellar granule
cell layer; a Normal, b Mild, c Moderate, d Severe. Sections are stained with haematoxylin
and eosin and photographed at magnification ×400
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Fig. 2.
a Effect of granule cell layer integrity grade on brain pH. Individual values and means ± SD
are shown for each histological grade. A significant difference was found between mild
grade and normal pathology (+
P
= 0.004); moderate grade and normal pathology (^
P
<
0.0001); severe grade and normal pathology (*
P
<0.0001) and severe and mild grades (#
P
=
0.0050). b Effect of granule cell layer integrity grade on the RNA Integrity Number (RIN).
Individual values and mean ± SD are shown for each histological grade. A significant
difference was found between moderate grade and normal pathology (+
P
= 0.0027) and
between severe grade and each of the other grades (*
P
< 0.0001 vs. normal pathology; #
P
=
0.0001 vs. mild grade; ^
P
< 0.0001 vs. moderate grade)
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Table 1
Demographic details and mean PMI, mean brain pH and mean RIN of clinical classification groups
Classification (N) Sex (N), Mean age (y) PMI (sd) Brain pH (sd) RIN (sd)
Alcohol abuse disorders 22 F (7), 55
M (15), 56 36 (16) 6.50 (0.32) 6.80 (1.89)
Control 34 F (8), 73
M (26), 61 30 (15) 6.51 (0.30) 7.07 (1.55)
Motor neurone disease 14 F (5), 67
M (4), 68 16 (11) 6.59 (0.27) 7.22 (0.86)
Multiple sclerosis 9 F (5), 58
M (4), 56 18 (12) 6.27 (0.33) 6.37 (0.98)
Psychiatric disorders 12 F (5), 52
M (7), 57 41 (18) 6.65 (0.22) 7.33 (1.03)
Other 14 F (8), 57
M (6), 60 44 (17) 6.60 (0.23) 7.28 (0.68)
Other—unconfirmed clinical diagnosis,
PMI
post mortem interval,
RIN
RNA integrity number
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Table 2
Mean brain pH, mean RIN, and mean PMI for granule cell layer grading groups
Granule cell layer grade N PMI, h (sd) Brain pH (sd) RIN (sd)
Normal 91 33 (18) 6.60 (0.23) 7.30 (0.90)
Mild 6 24 (17) 6.24 (0.27) 6.60 (0.74)
Moderate 5 23 (11) 6.05 (0.22) 5.80 (0.29)
Severe 3 18 (4) 5.77 (0.15) 1.60 (2.77)
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