Rapid estimates of neuron number in the confocal microscope combined with in situ hybridisation and immunocytochemistry

Brain Research Protocols 8 (2001) 113–125 www.elsevier.com / locate / bres

Protocol

Rapid estimates of neuron number in the confocal microscope combined with in situ hybridisation and immunocytochemistry I.P. Johnson* Department of Anatomy and Developmental Biology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2 PF, UK Accepted 3 May 2001

Abstract A rapid counting protocol is described which combines the optical disector and Cavalieri methods on non-embedded slices of fixed tissue viewed in the confocal laser scanning microscope. By eliminating the embedding stage and avoiding the need to align adjacent sections in the z plane for counting, considerable time savings are gained over physical disector methods. It also allows the remaining non-embedded sections to be used for other purposes, such as in situ hybridisation and immunocytochemistry. Starting with fixed brainstem, it was possible in less than 2 h to determine the total number of motoneurons in both facial nuclei of an adult Sprague–Dawley rat. This method revealed that the normal facial nucleus contained approximately 3200 motoneurons (n512 rats). One month following facial nerve avulsion (n54 rats), mean numbers of motoneurons were reduced by 75%. Using intervening sections, changes in neuronal number were compared with changes in in situ hybridisation signal and immunostaining.  2001 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing and imaging techniques Keywords: Neuron counting; Vibratome; Confocal microscope; Disector

1. Type of research

2. Time required

Methods of tissue preparation optimised for cell counting, or for cytochemical staining (e.g., in situ hybridisation or immunocytochemistry) can often be mutually exclusive, necessitating the duplication of experiments. The present report describes a protocol that enables rapid sampling of regions of the brainstem for unbiased cell counting and tissue staining techniques such as in situ hybridisation and immunocytochemistry. Advantages include:

2.1. Entire experiment

• • • •

Efficient sampling. Multiple analyses per tissue sample. Fewer experimental replicates. Ability to store free floating sections for up to 3 months prior to in situ hybridisation.

The method should also be suitable for analysis of non-neural tissues. *Tel.: 144-20-7794-0500; fax: 144-20-7830-2917. E-mail address: [email protected] (I.P. Johnson).

• 1 h 35 min for cell counts per rat. • Add 3 days for in situ hybridisation. • Add 1.5 days for immunocytochemistry of specimens obtained from up to six rats.

2.2. Motoneuron counts after facial nerve avulsion • Facial nerve avulsion surgery: 15 min / rat. • Perfusion–fixation, removal and trimming of brainstem: 20 min / rat. • Vibratome serial sectioning of brainstem: 30 min / rat. • Identification of facial nucleus within vibratome series: 10 min / rat. • Selection of sample of facial nucleus sections (n55 approx.), staining with the fluorescent dye YOYO-1 iodide (YOYO) for motoneuron counts, mounting and cover slipping: 10 min / rat.

1385-299X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S1385-299X( 01 )00079-4

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• Motoneuron counting in confocal microscope: 1 h / rat. • Data entry and spreadsheet calculation of total number of motoneurons: 10 min / rat.

2.3. Immunocytochemistry and in situ hybridisation • Surgery, perfusion and selection of sections as above: 1 h 30 min / rat. • Immunocytochemistry / lectin staining (including overnight incubation in primary antibody or lectin at 48C): 24 h (1–6 rats, data not shown). • Non-isotopic in situ hybridisation: 2–3 days (1–6 rats, data not shown).

250 ml saline followed by approximately 500 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Both solutions were at room temperature. The brainstem was removed and left for 4–24 h in the same fixative at 48C. Using a razor blade, a small ‘V’-shaped nick was made in the posterolateral part of the left side of the brainstem to allow the subsequent orientation of sections. A dissection microscope helps, but is not essential for this part. The brainstem was then trimmed so that the 1.5 mm long region containing the facial nuclei (which were to be counted) lay roughly midway in a piece of tissue 3–4 mm in rostro-caudal length.

4.2. Vibratome sectioning (one brainstem515 min) 3. Materials

3.1. Equipment • Vibratome (Oxford Instruments, UK). • Bright field microscope (preferably inverted microscope or dissection microscope with transillumination). • Image analysis system (Kontron) • Confocal laser scanning microscope (Bio-Rad MRC 600 or similar). • Multiwell chamber (48-well cell culture cluster; Costar, Cambridge, MA, USA).

3.2. Chemicals and reagents • 4% paraformaldehyde in 0.1 M sodium phosphate buffer. • YOYO-1 iodide fluorescent stain for nucleic acids (Molecular Probes, Eugene, OR, USA; catalog No. Y-3601). • Cyanoacrylate glue, fine paintbrush, 0.1 M sodium phosphate buffer, mountant suitable for fluorescence microscopy (e.g., Citifluor, Agar Scientific, Essex, UK), glass microscope slides and coverslips.

4. Detailed procedure

4.1. Animals, surgery, perfusion and tissue trimming Twenty adult (12-week-old) Sprague–Dawley rats were used. They were kept under a standard 12-h on / off lighting regime and fed ad libitum. The right facial nerve was injured in eight rats and 12 rats served as non-operated controls. As a check on the reproducibility of the method, tissue from the control rats was processed in two separate experiments (n56 rats for each experiment). One month after surgery (see legend to Table 2 for details), all rats were deeply anaesthetized (60 mg / kg sodium pentobarbitone i.p.) then perfused transcardially with approximately

Surface fixative was removed with filter paper and the specimen was fixed to the specimen stage of a vibrating blade tissue sectioner (Vibratome series 1000; Microfield Scientific, Oxford, UK) using Cyanoacrylate glue. With the specimen submerged in phosphate buffer, serial 70 mm sections were cut and each section transferred using a paintbrush to individual phosphate buffer-filled wells of a Multiwell chamber (48-well cell culture cluster; Costar). Sectioning was continued until only about 0.5 mm of specimen was left attached to the specimen stage of the vibratome. Serial sectioning the whole specimen in this way followed by identification of those sections containing the facial nucleus was found to be much more efficient than repeated examination of sections as they were cut. It also prevented the loss of important sections, as generally happened when sample sections were taken blindly at larger intervals (e.g., 200 mm) through the tissue block.

4.3. Location of section series containing the facial nucleus (one brainstem55 min) The Multiwell chamber usually contained a total of 35–40 sections of 70 mm thickness (i.e., a 2.5–3.0 mm length of brainstem). The length of brainstem finally sectioned was always shorter than the 3–4 mm length of brainstem glued to the specimen stage, since tissue from the rostral surface was invariably lost as incomplete fragments at the commencement of sectioning, and up to 0.5 mm of unusable glue-impregnated tissue was usually left attached to the specimen stage. The sections in the Multiwell dish were examined with a bright field microscope using 34 objectives and 310 eyepieces. An inverted microscope is preferable, as most are designed to take Multiwell dishes. However, an upright student teaching microscope with the condenser out and the specimen stage racked down will also suffice. Those sections containing the facial nucleus were readily distinguished in this way (Fig. 1). The wells containing sections corresponding to the beginning and end of the section series containing this nucleus were recorded by making a mark near the appropriate wells with a fibre-tip pen. Identification of the

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Fig. 1. One of a series of 70 mm thick vibratome sections taken serially through fixed rat brainstem and viewed unstained at low magnification while free-floating in PBS in a Multiwell dish. Identification of the section series containing the facial nucleus (arrow) is easily and rapidly achieved. From this section series, a smaller number of sections (e.g., every fifth section starting at a random number between 1 and 5) can be selected randomly and systematically for stereology or staining. The nick seen in the posterior portion of this section identifies it as the left (non-operated) side of the brainstem.

section series through the facial nucleus is done most efficiently by starting the visual examination in the middle of the entire group of 35–40 sections, as this is where the facial nucleus is most likely to be large and prominent (|1 mm in diameter). Adjacent sections can then be examined to trace the nucleus to its most rostral and caudal poles. The last section in the series through the nucleus typically contained around five large motoneurons. Fortunately for this procedure, the facial nucleus tapers abruptly at its poles, so that it transforms from a large unequivocally identified structure to nothing in the space of 1–2 sections. In this study, approximately 22–25 sections contained the facial nucleus in adult rats, giving a facial nucleus length of 1500–1750 mm. With a section thickness of 70 mm, the estimated error in determining the total number of sections through the facial nucleus is therefore 70–140 mm, or less than 10%. Application of this method of analysis to less well-defined central nervous system (CNS) areas may require the use of other morphological or topographical criteria to define the sampling area, or the use of immunocytochemical or retrograde labelling methods. In our hands, Vibratome sections are well suited for retrograde labelling studies [1–3]. Sections can be used for immunocytochemistry [4], lectin histochemistry [7] or in situ hybridisation [12] within 1 day of cutting (sections stored in buffer at 48C). If the sections are first dehydrated in graded ethanols to absolute ethanol and stored at 2208C,

they can also be used after 7 days (immunocytochemistry) or after 3 months (in situ hybridisation) storage. Other cryoprotectants, such as mixtures of ethylene glycol and sucrose in buffer, may also be suitable, but have not been tried here.

4.4. Systematic random sampling and fluorescent staining (one brainstem515 min) Starting with a random number from 1 to 5, every fifth section from this series was taken and placed carefully using a paintbrush on a microscope slide. Very occasionally, a section in the series was lost or could not be used because of technical problems. In this case an adjacent section was used and then sampling reverted to the original interval. Decisions on sampling intervals are probably best made in a pilot experiment, as they will depend on the total number of sections through the nucleus and the number of neurons typically present in one of the sections. We started with every third section (a guess) and increased this to every fifth section, as we found increasing in sampling interval speeded up the analysis yet made less than 10% difference to our estimates of neuron number. Using every fifth section as our interval, a total of 4–5 sections were removed from the Multiwell dish using a fine paintbrush and arranged linearly on a single microscope slide. The absolute length of this row of sections (|2.5 cm here) will

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be determined by the size of slide and coverslip available. After removing excess buffer from around the sections, they were covered with a few drops of a 1:1000 dilution (in 0.1 M phosphate buffer) of the fluorescent dye YOYO1 iodide (Molecular Probes, catalog No. Y3601). This dimeric cyanine dye for nucleic acids was selected as it gave the fluorescent equivalent of a conventional Nisslstained section (Fig. 2), facilitating the identification of brainstem nuclei and of cellular elements for subsequent confocal laser scanning microscopy. Where a correspondence between YOYO staining and Nissl staining is called into question, this can be checked by staining intervening sections with a conventional Nissl stain. It is also possible, due to the thick nature of the sections, to compare the YOYO stained epifluorescence image at various objective magnifications directly with that obtained by transillumination of the same section with white light. In the latter case, closure of the iris diaphragm to increase interference enables cell bodies and nuclei to be visualized with ease. After 1–2 min, and taking no precautions to avoid exposure to the fluorescent strip lighting in the laboratory, excess YOYO was removed from the slides with filter paper, taking care not to touch the sections. A small amount of mountant (Citifluor, Agar Scientific) was placed on the sections and all 4–5 sections coverslipped. It is advisable to optimise YOYO staining on ‘spare’ sections outside the region of interest, using the above concentration and time of staining as a guide, and adjusting one or other parameter depending on the intensity of cellular fluorescent staining and the amount of background fluorescence seen. Testing YOYO staining in this way will add 15–30 min to the procedure, but can avoid the need subsequently to remove coverslips and re-stain all the sections in a series from a group of several animals. Where slides were not to be examined immediately, they were left in the dark overnight at room temperature and the edges of the coverslip then sealed with nail varnish. Slides prepared in this way have been stored for 18 months in the dark at 48C without noticeable loss of fluorescence.

4.5. Determination of mean area of facial nucleus (one brainstem510 min) This was done using the Cavalieri method [5]. Each (YOYO-stained) section on the slide was viewed by transillumination (not epi-illumination), with the condenser lens out and aperture of iris diaphragm slightly too small so as to create a small amount of interference and improve visualization of the tissue slice. The facial nucleus was clearly visible on the basis of its location in the brainstem and the morphology of its neurons (cf. Fig. 1). Using the 310 objective to encompass the facial nucleus on one side of the brainstem, the image was captured using a charge coupled device (CCD) camera and displayed as a monochrome image on a video display unit (VDU), so that the facial nucleus on the screen was seen as an ellipsoid area

of approximately 60340 mm. Using the interactive software of an image analysis system (Kontron) the boundaries of this nucleus were drawn on screen, taking care to join facial motoneurons to their nearest neighbour, and the calibrated area enclosed by this boundary determined. This was done for each facial nucleus profile in each of the 4–5 sections from each rat.

4.6. Calculation of V (ref ) The mean area of the facial nucleus for each rat was then calculated (a). This was multiplied by the total number of sections through the facial nucleus (s) and then multiplied by the section thickness (t) to give an estimate of the volume of the facial nucleus, V (ref), where V (ref)5ats.

4.7. Optical disector (steps 4.7 – 4.11 for one brainstem560 min) Each section on the slide was viewed in the CLSM (Bio-Rad MRC 600), using 488 nm laser illumination, a 3–4 mm pinhole aperture and a 320, 0.7 numerical aperture dry objective (Olympus D Plan Apo 20). This gave an optical section thickness of less than 5 mm, which was half the distance between optical sections used for the disector counts, ensuring that the optical disectors did not overlap. In most cases, the facial nucleus was sampled at four slightly overlapping areas in the x–y plane by moving the slide so that the edge of the field of view was adjacent to either the anterior (A), posterior (P), medial (M) or lateral (L) borders of the nucleus (Fig. 3). Near the poles of the facial nucleus, the area of the nucleus was smaller and so sampling was modified to three, two or even one field, depending on the size of the nucleus. This approach, which sampled most of the area of the facial nucleus in a section, was used to increase the probability that all the motoneurons in the section through the facial nucleus had an equal chance of being counted. Sampling smaller areas of the nucleus would have been more efficient, but may have introduced error due to the non-uniform distribution of motoneurons (e.g., Fig. 2). Within the sampling fields, the most superficial (top) border of the vibratome section was identified by its irregular contour, producing a fragmented ill-structured image on the VDU. To find the top of the section quickly, the section was brought into focus by eye using epifluorescence. This focal plane tended to be in the middle of the section. Continuous confocal scans were then made using a 5 mm Z-step increment so that subsequent scans were coming out of the section. Automatic scanning was halted when the image first disappeared manual scans were then made back down into the section in 1–2 mm increments until the (usually fragmented) image first started to appear. This was taken as the top of the section and a mental note made of the z-coordinate. Resetting the z-coordinate to zero was found to be

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Fig. 2. (a) A 70 mm thick vibratome section of fixed rat brainstem 1 month following facial nerve avulsion, stained briefly with the fluorescent dye YOYO-1 iodide and viewed using epifluorescence. The facial nucleus and surrounding topography are easily identified, enabling rapid alignment of pre-determined areas of the facial nucleus for subsequent confocal microscopy. (b) Enlargement of the operated facial nucleus from (a). Very few motoneurons are seen. The boundaries of the nucleus are still discernable as the junction between overall increased fluorescence (facial nucleus) and the lower overall fluorescence of the neuropil. (c) Enlargement of the non-operated facial nucleus from (a). Many motoneurons are seen. Note that the distribution of motoneuronal cell bodies within the nucleus is not homogeneous. This can affect the accuracy of estimates of motoneuron number if they are based on small sample volumes of the nucleus.

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Fig. 4. Appearance of the image as seen on the visual display monitor after superimposing two optical sections 10 mm apart taken through the lateral quadrant of the facial nucleus. Only the green nucleated cell bodies .20 mm in diameter are counted. Red cell bodies and those with a mixture of red and green / yellow were not counted. In this study, classification of cell bodies as green (vs. green / yellow) was much more efficiently done directly on the VDU than on prints, since additional colours were often introduced into the latter as a result of poor colour balancing of the printer.

unnecessary and time-consuming. From this point at the top of the section, the optical plane was stepped down 30 mm into the section. A scan was taken at this level (look-up section) and stored as a temporary file.

4.8. Optical disector continued A second scan (reference section) was then taken 10 mm

deeper into the section, without altering the x–y co-ordinates.

4.9. Optical disector continued The second scan (reference section) was left on the screen and was merged with the stored first scan (look up section). The MRC 600 CLSM automatically assigns different colours to these two scans (green and red,

Fig. 3. Schematic representation of the basic protocol. The brainstem of approximately 3 mm height is shown to contain bilaterally the nuclei or areas of interest (small gray ovals). Serial vibratome sectioning of the brainstem at 70 mm produces about 30 sections within which the section series containing the nucleus of interest can be defined. Sections are taken randomly and systematically for analysis in the confocal microscope. Estimation of the number of ‘tops’ (Q 2) is done by superimposing two optical sections each 10 mm apart from the middle 10 mm of the section. In this study, where there is considerable anistropy of the neurons within the nucleus, a large area of the nucleus was sampled by determining Q 2 in four (sometimes overlapping) areas and then determining the mean value for Q 2 for the vibratome slice. Determination of Q 2 is facilitated by storing the two confocal scans as separate colours and then superimposing these on screen. Of the three neurons (defined here as having diameters .20 mm) containing a nucleus which appear when the two optical sections are superimposed, one is identified by its yellow colour as being present in both optical sections, one is identified by its red colour as being present in the lower optical section only and one is identified by its green / blue colour as being present in the upper section only. In the present protocol only the green / blue neuron is counted. The left and lower borders of the counting frame were taken as ‘forbidden lines’. Any profile which crossed these lines, or their extensions above and to the right of the counting frame, was not counted.

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respectively). After superimposing the two scans (Fig. 4) the number of green neuronal profiles .20 mm in diameter displaying a nucleus (‘tops’) were counted directly on the screen. Neurons were defined as nucleated cells .20 mm diameter with moderate to large cytoplasmic areas containing Nissl bodies. Configuring the display to include a 20 mm scale bar can help in this respect. Neurons were only counted on the screen if they were contained within the rectangular boundary that was displayed with each scan and if they did not cross ‘forbidden lines’ [6], in this case the left- or lower borders of the sampling ‘brick’. Where necessary, these forbidden lines were extended linearly on the screen beyond the boundaries of the counting frame. To save time and disc space, subsequent optical scans can be made to overwrite the previous one. However, this can be dangerous when developing the method or introducing new investigators, as it precludes any retrospective analyses or checks. Thus, on one occasion, it was found to be very useful to have saved the two scans using a WORM drive when it became clear that one investigator’s criteria for defining a motoneuron cell body differed markedly from that the other three investigators, and a re-count was needed after the consensus criteria were agreed. It was also found to be more efficient simply to write down the number of tops counted on the VDU, rather than switch to a data base program and enter these, as the scans were made. Small yellow areas could occasionally be seen on the processes of green neurons. These areas did not represent inclusion of the motoneuron in both optical sections. Instead, the small regular shape of most of these yellow areas indicated that they belonged to the nuclei of neuroglial cells in the look up section. The presence of a nucleus surrounded by a very small amount of cytoplasm (,20 mm diameter) together with a lack of Nissl-type staining allowed a distinction to be made between neuroglia and neurons.

greater than 50%, a single scan was taken in the centre of the facial nucleus. Where ‘tops’ were counted in .1 area of the facial nucleus, the mean number of ‘tops’ (M) for that particular section of the facial nucleus was calculated. Where the area facial nucleus was small enough to be encompassed by a scan of a single area, then the number of ‘tops’ in this single area was taken as M. Thus for each section through the facial nucleus, a single value for M was obtained.

4.12. Optical disector continued Repeat stages 4.7–4.11 for all YOYO-stained sections per rat.

4.13. Calculation 1 Where n5number of YOYO-stained sections per rat, and M5mean number of ‘tops’ per facial nucleus, calculate mean number of ‘tops’ (Q 2) per rat: Q 2 5 sum M1 . . . Mn /n

4.14. Calculation 2 Where a (dis)5area of disector and t5section thickness, calculate disector volume, V (dis): V (dis) 5 a (dis) ? t In this study V (dis)5200 934 mm 2 ?10 mm52.0093?10 3 mm 3 .

4.15. Calculation 3 Calculate the number of neurons per unit volume of the disector (Nv):

4.10. Optical disector continued

Nv 5 Q 2 /V (dis)

The mean number of tops for each nucleus, based on up to four sample areas, was determined (Q 2). With the confocal microscope pinhole aperture set at 3–5 mm, the numerical aperture of the objective chosen reduced the depth of field to less than 5 mm, and for the particle that was counted (nucleated cell body .20 mm diameter), this ensured that there was no overlap of optical sections (10 mm apart).

4.16. Calculation 4

4.11. Optical disector continued Where the facial nucleus was large enough, stages 4.7– 4.11 were repeated for the medial, anterior and posterior quadrants of the nucleus. Where the facial nucleus was smaller, it was often possible to obtain images for only three or two such areas with less than 50% overlap. Where the overlap of adjacent images in the x–y plane was

Having calculated the volume of the facial nucleus [V (ref)] using the Cavalieri method (see above), calculate the number of facial motoneurons (N): N 5 Nv ?V (ref) Data were entered into a spreadsheet (Table 1) for each animal. These spreadsheets could then be linked to compile summary data and calculate standard errors and deviations of the mean.

4.17. Calibration of section thickness Using a micrometer gauge, the thickness of a cork disc was measured (C). A non-essential region of fixed brain-

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Table 1 Example of spreadsheet used to calculate neuronal number Code: normal 6 OP Section

Nucleus area

1 2 3 4 5 6

Section thickness

429 061 709 359 1 014 269 857 634 174 876

Mean

860 420.7

Disector area Disector height Disector volume Total No. sections thro nucleus Total volume of nucleus Nucleus volume / disector volume Total No. of neurons

70 ] 70 ] 70 ] 70 ] 70 ]

30 034 270 49 655 130 70 998 830 60 034 380 12 241 320

70 ]

44 592 786

10 ] 2 009 340

Disector data, tops (Q 21 )

Volume

M

L

A

P

Mean

7 1 5 5 5

8 7 6 9 6

5 5 4 10 5

5 9 5 9 5

6.25 5.5 5 8.25 5.25

6.05

200 934 ]]

24 1.07E109 532.6261 3222.388

Data entered separately into the spreadsheet is shown in bold type. Other data is pre-existing, either as formulae (ordinary type) or as values that remain constant throughout the experiment (italic, underlined). By using a standard format, many spreadsheets can be linked to derive sample means and other statistical parameters.

stem was trimmed so that it was approximately 3–4 mm in height and the top and bottom edges were cleanly cut and parallel to each other. The brainstem was glued to the cork disc and the combined height of the tissue plus disc was measured (T ). The height of the specimen (h) is given by (T2C). The specimen was then sectioned from top to bottom until the first signs of cork appeared and note made of the total number of sections taken (n). The calibrated section thickness is calculated as h /n.

with positions of n11 . . . n14 in the section series through the facial nucleus, n being the random starting point for sections used for neuron counts. In this protocol additional sections have been used successfully for nonisotopic in situ hybridisation, immunocytochemistry and lectin histochemistry (data not shown).

5. Results

4.18. Additional analyses of remaining sections This will vary according to the type of experiment undertaken. The protocol allows sections for these additional analyses to be taken randomly and systematically

Starting with fixed brainstem, the total number of facial motoneurons was determined bilaterally in 20 rats in approximately 33 h. Remaining sections were available for a variety of additional analyses, including in situ hybridisa-

Table 2 Total numbers of numbers in the left and right facial nuclei of adult Sprague–Dawley rats Normal

Rat Rat Rat Rat Rat Rat

1 2 3 4 5 6

Mean S.D.

1 m Crush

1 m Avulsion

Right

Left

Operated (R)

Non-operated (L)

Operated (R)

Non-operated (L)

3676 3622 3631 3666 3305 3222

3404 3118 3385 3233 3090 3307

3014 2889 2903 3083

3619 3404 3314 3523

884 889 719 733

3323 3372 3397 3023

3520.33 201.6955

3256.17 132.872

2972.25 92.6188426

3465 133.6687

806.25 92.863251

3278.75 173.2481

Non-operated rats (n56) have approximately 3388 motoneurons. One month following facial nerve crush, motoneuron numbers are reduced by 14% compared to the non-operated nucleus, whereas 1 month following facial nerve avulsion, motoneuron numbers are reduced by 75% compared to the non-operated nucleus. Operated rats were anaesthetized (2% halothane carried by 2 l O 2 per h) and the right facial nerve either (i) crushed (n54) or (ii) avulsed (n54) as it emerged from the stylomastoid foramen. Skin was sutured, analgesic given (subcutaneous bupranorphine) and animals allowed to recover for 1 month before perfusion–fixation.

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122 Table 3 Reproducibility of the method Normal

Rat Rat Rat Rat Rat Rat

1 2 3 4 5 6

Mean S.D.

Right

Left

3976 3163 2668 4182 2061 3404

3561 2985 3331 2519 2532 2816

3242.42 797.3804

2957.24 423.7512

Total numbers of numbers in the left and right facial nuclei of a second group adult Sprague–Dawley rats. These non-operated rats (n56) have approximately 3099 motoneurons in the facial nucleus, which is 9% less than the estimate of 3388 for the first group (see Table 1).

tion and immunocytochemistry, with the result that fewer animals were needed for the experiment.

5.1. Normal nucleus and effects of facial nerve avulsion When values for the left and right facial nuclei were combined for all six normal rats in the first non-operated group, a mean of 3388 motoneurons in the facial nucleus was calculated (Table 2). When the experiment was repeated with the second group of six normal rats, estimates of motoneuron number were 9% lower at 3100 (Table 3). None of the differences between the two groups of normal rats were statistically significant (Mann–Whitney U-test). One month following nerve crush, there was a slight (14%) reduction in motoneuron numbers on the operated side compared to the non-operated side. This reduction was not apparent on qualitative examination of the sections and it was not significantly different to values obtained for the facial nuclei of non-operated rats. One month following nerve avulsion, however, there was a clear qualitative loss of motoneurons on the operated side (Table 2, Fig. 2), which amounted to a 75% loss.

5.2. Staining of additional sections Immunostaining for calcitoxin gene related peptide (CGRP) and glial fibrillary acidic protein (GFAP) was clear in sections processed immediately after cutting or after storage overnight in phosphate-buffered saline (PBS) at 48C (data not shown). CGRP and GFAP immunostaining was also clear after storage of sections in methanol at 2708C for 7 days but lost after storage for 1 month. The effect of other cryoprotectants, such as glycerol, on immunostaining has not been assessed. In contrast to the short preservation of antigenicity within the sections, clear in situ hybridisation for GAP-43 mRNA was seen after 1–3 months storage in methanol at 2708C (data not shown). In the present study, increased GFAP and GAP-43 expression characterized the injured nucleus [12]. It was

also possible to stain the vibratome sections with conventional aqueous Nissl stains (data not shown). Care, however, needs to be taken to avoid over staining. This can be done by using dilute Nissl stain initially and de-staining in alcohols to produce a section which when mounted wet on a slide has an overall very light blue appearance.

6. Discussion Accurate determination of neuron numbers is crucial in many studies of development, ageing, injury and disease. While counting every neuron in the area of interest would suffice, this is usually prohibitively time-consuming. As a result, estimates of total neuron number are instead usually based on counts made on sample sections taken from the whole area of interest. Earlier sampling methods made explicit assumptions about the size and shape of neurons with respect to section thickness, and implicit assumptions about the sample sections being taken systematically yet randomly from the whole area of interest [13–15]. If these assumptions were incorrect, errors due to bias would be introduced into the estimates obtained. As the limitations of these older methods have been recognized, they have now largely been superseded by so-called unbiased stereological methods which are in theory independent of changes in size and shape of the objects being counted [16–18] These methods rely on a comparison of profiles seen in one section with those seen in a second section. Such assumption-free stereological methods for estimating total neuron number are becoming increasingly common [19–23]. It is unlikely that these methods are totally unbiased, since at some point, they usually involve some unverified pragmatic assumptions (e.g., that all objects have an equally chance of being sampled). Nevertheless they come closer to providing unbiased estimates of particle number than previous sampling methods. One drawback of assumption-free methods is that they can be very time-consuming because it is usually necessary to cut many serial tissue sections and it is often very difficult to align adjacent sections accurately in the z plane to allow ‘tops’ to be counted. In previous studies, many serial sections have been cut physically (physical disector) and images either superimposed using photographic methods [24] or a drawing tube [25]. Both methods are laborious with the possibility of misalignment of adjacent sections in the z plane. Here, a method is described which eliminates alignment error in the z plane and allows rapid sampling from large volumes of tissue for stereological counts of cells. While the use of optical disectors is not new [26,27], the method described here allows massive time savings to be made, and also allows intervening sections to be used for other forms of tissue processing, such as in situ hybridisation and immunocytochemistry. The method should be directly applicable to most areas of the CNS where neurons are gathered together in discrete nuclei. It could also be adapted to enable unbiased estimates of

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discrete populations of neurons labelled using fluorescent markers (e.g., in combination with in situ hybridisation, immunocytochemistry or retrograde axonal transport methods). The use of specific neuron markers would facilitate the analysis of sub-populations of neurons or collections of neurons that are not gathered together in morphologically discrete nuclei. It is also possible that estimates of numbers of non-neuronal cells could also be made if suitable morphological or labelling criteria could be defined.

6.1. Trouble shooting 6.1.1. Sectioning and section thickness Persevering with a tissue block which fails to produce useable serial sections within the first 10 traverses over the vibrating blade is fruitless; sections emerge fragmented, scored, wedge-shaped, or not at all, and all subsequent analysis is unreliable or impossible. If this problem occurred it was found best to remove the specimen by sliding a razor blade between it and the specimen support. Any ragged edges were trimmed from the specimen, if possible a small slice was taken from the top or bottom of the specimen to reduce its overall height and if the specimen appeared too soft it was post-fixed for a further 24 h at 48C before remounting. If this failed, we found it more efficient to simply abandon the tissue block for quantitative purposes. Of course it may not be practicable to discard some valuable tissue samples. In this case, the block may be processed for analysis using different types of section (e.g., cryoprotected and frozen, or embedded in paraffin wax). Alternatively, if vibratome sections are essential the variable number of useful sections eventually obtained could be used for non-quantitative analyses. Care needs to be taken to decide on an appropriate vibratome section thickness. This needs to be thick enough to reduce the time spent on serial sectioning and to allow serial confocal scans to be made of it. However, the section must not be so thick that the area of interest (e.g., facial nucleus) cannot be discerned on wet sections using bright field microscopy, nor so thick that antibodies, probes and other staining reagents cannot penetrate. It was found here that a section thickness range of 50–100 mm was compatible with all the above requirements. In general, it was found that vibratome sections .50 mm can be cut with fewer technical problems, including lost sections, than sections ,50 mm. There will be an upper limit to section thickness beyond which there is too much attenuation of the laser beam of the confocal microscope to produce reasonable images. We find that section thicknesses up 100 mm still result in good image quality for rat brainstem, but this is likely to vary for different tissues. A common sectioning problem, characterized by the appearance of sections which are alternately too thick and then absent, is due to the specimen bending as the vibratome blade reaches it. This can occur if the specimen height is greater than the diameter of its base, if the specimen is only loosely glued to the platform, or if the specimen is too soft (weak / poor

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fixation). The first two are easily remedied. Where using a higher concentration of fixative and overnight post fixation is incompatible with subsequent immunocytochemistry, it is possible to support soft tissue by embedding it in 5% agar before cutting. Cooling the stage and using ice cold buffer in the bath of the vibratome may also help, as may reducing the blade advance speed and increasing the amplitude of the blade vibration. Investigators may also discover empirically that certain blades, e.g., halved domestic razor blades, disposable microtome blades, etc., have sharpness and tensile properties which suite them for cutting certain types of tissue. Care should be taken to remove any oil from these blades with alcohol before use. Failure of tissue to section correctly can also be due to excess glue creeping up the sides of the specimen after it has been submerged. To avoid this, use only a small drop of glue and spread it as a thin film using a filter paper spreader before applying the specimen and leave the tissue for approximately 30 s in air to stick fast before submerging in buffer. In some cases, the sections near the beginning or end of the section series showed the facial nucleus on one side only which in normal animals was probably due to non-orthogonal sectioning of the brainstem. Strictly speaking, sampling of sections from the series would have to be undertaken separately on the left and right sides. However, we found that using the first section in the series that contained both nuclei for sampling gave very similar values for estimates of neuron number.

6.1.2. Mounting medium, fluorescence and refraction As most confocal microscopy will rely on excitation of fluorochromes by the laser beam, it is important that a mountant is chosen which retards photo bleaching. Another consideration is to match the refractive index of the mountant with that of the coverslip. Failure to do this will result in refraction of the emitted light from the specimen as it passes through the mountant and the coverslip. This can lead to measurement errors in the z plane. In theory, the objective lens should also be immersed in a medium of the same refractive index as the coverslip and mounting medium to avoid refraction errors in z plane measurement [8]. This is possible for permanently mounted specimens using 340, 60 and 100 oil immersion lenses with matching condensers, but the use of such magnification reduces the area of tissue able to be sampled by the disector. Sampling of such a small area may not pose a problem for regions such as the hippocampus [9]. However, for anisotropic structures such as the facial nuclei, it was considered safer in this study to sample a large area of the facial nucleus seen in individual sections. For efficiency, this was done using a dry 320 objective. One possible, but more time-consuming solution would be to use high numerical immersion lenses to construct a shallow depth of field montage of the section at two focal planes. This is possible using commerciallyavailable software linked to the x–y controls of the

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microscope specimen stage. It is unlikely that the precise depth of the first optical section in this study was 30 mm from the top of the vibratome slice, as there would have been refraction of the beam at several interfaces (specimen, aqueous mountant, coverslip and air) before it entered the objective lens. The operating software of some confocal microscopes allows best guesses for the correction factors for some of these interfaces to be entered and measured dimensions are adjusted accordingly. The extent to which such correction might allow measurement of the true z displacement was not tested in this study, since our uncorrected measures still gave values for total motoneuron number that were within the ranges reported by others using unbiased stereological methods. To avoid the possibility that the optical section thickness overlaps with that of the optical disector interval, it is important to keep the pinhole aperture as small as possible. It has been calculated for a 325, 0.8 NA dry objective in the Bio-Rad MRC 600 CLSM that a closed (1 mm) pinhole aperture will give an optical section thickness of 1.4 mm, whereas an open pinhole (7 mm) will give an optical section thickness of 7.8 mm [10]. In this study we used a pinhole aperture of 3–4 mm, with an estimated optical slice thickness of 5 mm. The compressive effect of the glass coverslip on the section may also affect estimates of neuron number. However, this effect appears to be slight, as we were unable to detect any significant change in the area of sections after cover slipping compared to when they were viewed free floating in buffer. A further measure of the degree of section compression could be obtained by measuring section thickness with a microcater before and after applying the coverslip. Alternatively, it might be possible to substitute objective lens immersion oil with viscous embedding media and avoid cover slipping altogether [11].

6.1.3. Definition of neurons for counting The definition of a cell profile in the CNS as ‘neuronal’ relies heavily on morphological criteria such as size, shape and pattern of staining. In this and other stereological studies where these criteria are used to define neurons for counting, errors will be introduced if experimental procedures alter size, shape or staining. Ideally, the feature used to define a neuron for counting should be unaffected by the experimental procedure, but this can prove difficult to control for in practice. Perhaps a compromise is to employ several inclusion criteria in the hope that some of these will remain unaffected by the experimental procedure.

3.

4.

5.

6. 7.

8. 9.

10.

11.

12. 13.

14. 15.

7. Quick procedure 1. Obtain 4% paraformaldehyde-fixed brainstem. 2. Cut serial 70 mm vibratome sections of brainstem

16. 17.

under buffer and place each section in a buffer-filled well in a Multiwell chamber. Section thickness5t. Identify section series containing facial nucleus by viewing wet sections in wells using low-magnification (e.g., 34) bright field microscopy. Number of sections through structure of interest (in this case the facial nucleus)5s. Starting at a random number between 1 and 5, stain every fifth section in the series through the facial nucleus with the fluorescent cyanine dye YOYO-1 iodide. Embed in Citifluor and coverslip. Remaining sections can be used for other staining methods including in situ hybridisation and immunocytochemistry. Using bright field microscopy and interactive image analysis software determine the area of the facial nucleus in each of the YOYO-stained sections per rat. Mean area of facial nucleus for all the sections in the sample5a. Calculate volume of facial nucleus, V (ref), where V (ref)5ats. Take a confocal scan (320 objective, 0.7 NA) of the lateral quadrant of the facial nucleus in a YOYOstained section, 20 mm from the top of the section and store the image (look-up section). Without altering the x–y co-ordinates, take a second confocal scan 10 mm deeper to the first scan. Superimpose second scan (reference section) with the stored (look-up) scan, using contrasting colours (e.g., red for the first scan, green for the second scan). Record the number of green motoneurons (‘tops’) that do not cross the ‘forbidden lines’ of the counting area. These correspond to motoneurons that appear in the reference section but not in the look-up section. Where the facial nucleus is large enough, repeat stages 6–8 for the medial, anterior and posterior quadrants of the nucleus. Where the facial nucleus is smaller, it may be possible to obtain images for only three or two such areas with less than 50% overlap. Where the overlap of adjacent images in the x–y plane is greater than 50%, take a single scan in the centre of the facial nucleus. Calculate mean number of ‘tops’ per facial nucleus. Mean number of tops per facial nucleus5M. Repeat stages 6–9 for all YOYO-stained sections per rat. Where n5number of YOYO-stained sections per rat, calculate mean number of ‘tops’ (Q 2) per rat. Q 2 5 sum M12n /n Where a (dis)5area of disector, calculate disector volume, V (dis), where V (dis)5a (dis)?t. Calculate number of neurons in disector volume, Nv, where Nv5Q 2 /V (dis). Calculate number of neurons in facial nucleus (N), where N5Nv?V (ref). Use remaining sections (i.e., every second, third or fourth section) in the series for additional analyses

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(e.g., in situ hybridisation, immunocytochemistry, etc.).

8. Essential literature references Original papers: D.C. Sterio, The unbiased estimation of number and sizes of arbitrary particles using the disector. J. Microsc. 162 (1984) 203–231. M.J. West, L. Slomiaka, H.J.G. Gundersen, Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231 (1991) 482–497. Books: C.V. Howard, M.G. Reed, Unbiased stereology. Threedimensional measurement in microscopy, in: Microscopy Handbooks, BIOS Scientific, Oxford, 1998, p. 41. S.W. Paddock, An introduction to confocal imaging, in: S.W. Paddock, Confocal Microscopy Methods and Protocols. Humana Press, NJ, 1999, pp. 1–34. Reviews: M.J. West, Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci. 22 (1999) 51–61. T.M. Mayhew, H.J. Gundersen, If you assume, you can make an ass out of u and me: a decade of the disector for stereological counting of particles in 3D space. J. Anat. 188 (1996) 1–15. F.M. Benes, N. Lange, Two-dimensional versus threedimensional counting: a practical perspective, TINS 24 (2001) 11–17.

Acknowledgements Supported by The Royal Society.

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