The RhoA transcriptional program in pre-T cells

The GTPase RhoA is essential for the development of pre-T cells in the thymus. To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors. The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development. Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 (Rag2) null mice. Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription.
1
Introduction
The T cell antigen receptor (TCR) complex comprises variable α/β subunits that recognise peptide/major histocompatibility (MHC) complexes and invariant signal transduction subunits of the CD3 antigen. A key stage in T cell development in the thymus is the selection of cells that have successfully rearranged their TCR-β locus. This occurs in T cell precursors, which do not express of the MHC receptors, CD4 and CD8 (double negative (DN) thymocytes) [1]. The DN stages of intrathymic differentiation can be followed by the sequential pattern of expression of CD44 and CD25. T cell progenitors enter the thymus as CD44+CD25− cells (termed DN1); these then express CD25 (DN2) and begin to rearrange T-cell receptor β loci. Cells then lose CD44 expression and continue β chain rearrangements to completion (DN3). Cells that successfully rearrange their TCR-β locus will express a functional receptor complex known as the pre-TCR comprising a TCR-β chain, the p-Tα subunit and the signalling subunits of the CD3 antigen [2–4]. When the pre-TCR is expressed at the cell membrane it promotes cell survival and entry into the cell cycle [5]; cells downregulate CD25 and transit to the DN4 pre-T cell subset. DN4 T cells undergo proliferative expansion and differentiate to CD4+CD8+ double positive (DP) cells. These cells then undergo TCR α-chain gene rearrangements and upon expression of a functional α/β TCR complex are subjected to positive and negative selection to generate CD4+ or CD8+ single positive (SP) thymocytes [6–8].
Normal pre-T cell development requires the coordination of a complex program of gene transcription by signal transduction networks mediated by the pre-TCR complex and cytokine and stromal signals [9,5]. Mice deficient for the recombinase activating (RAG) genes which are unable to rearrange TCR-β subunits and express a pre-TCR are unable to progress beyond the DN3 stage of thymocyte development [10]. Similarly, the absence of pre-Tα or CD3 subunits blocks thymocyte development at the DN3 stage [11,3]. The transition of thymocytes beyond the pre-T cell stage of thymocyte differentiation is also dependent on signal transduction networks mediated by tyrosine and serine kinases [3,12,13]. In this context, crucial responses are mediated by Rho family guanine nucleotide binding proteins such as RhoA, Rac-1 and CDC42 [14–17].
The importance of RhoA for thymocyte development has been demonstrated by studies of transgenic mice that express Clostridium botulinum C3-transferase under the control of T cell specific promoters such as the p56lck and CD2 promoters [17,15,18]. This toxin selectively ADP-ribosylates RhoA within its effector-binding domain and abolishes its biological function. Transgenic mice that express C3-transferase under the control of the p56Lck promoter have a small thymus and severely reduced numbers of peripheral T cells [15,17]. This phenotype is caused by survival defects in DN2 and DN3 thymocytes that lack Rho function [15]. During embryogenesis no cells progress beyond the DN1 stage but in adult mice a few T cell progenitors survive and develop to DN4s and beyond [15]. This ‘leakiness’ either reflects selection of cells that compensate for loss of Rho function in DN2/3 thymocytes or reflects heterogeneous and asynchronous expression of the lck promoter in adult DN thymocytes. The few DP thymocytes and mature T cells found in adult Lck-C3 transgenic mice have numerous defects including reduced survival, proliferation, integrin mediated cell adhesion and defective cell motility [15,19]. A complementary strategy to probe Rho function in the thymus used the CD2 locus control region (LCR) to target C3 transferase to T cell progenitors (CD2–C3 mice) [18]. The inhibition of Rho function at the DN2/3 stage in CD2–C3 mice is not leaky and causes T cells to become blocked in differentiation at the DN3 stage of thymus development. CD2–C3 mice thus have a thymic phenotype indistinguishable from the phenotype of Recombinase gene null mice or mice lacking key structural or signaling components of preTCR complex [10,18,20,21]: thymocyte development is blocked at the pre-T cell/DN3 stage [18].
The basis for the failure of pre-T cell differentiation in CD2–C3 transferase mice is not known. One way to address this issue is to use microarray gene expression profiling to determine the impact of losing Rho function on transcriptional responses in pre-T cells. Previous studies in transformed cell lines have identified a role for RhoA in regulating activating protein-1 (AP1) family of transcription factors [22–24] but transcriptional targets for RhoA signal transduction in primary non-transformed cells have not been explored. The present data show that RhoA regulates expression of genes encoding members of the Fos/Jun and early growth response (Egr) family of transcription factors but also has an impact on expression of genes regulating diverse biological functions including serine/threonine kinases, protein phosphatases, enzymes that regulate protein biosynthesis and proteins that regulate nuclear structure and function.
2
Materials and methods
2.1
Mice
Mice were bred and maintained under specific pathogen-free conditions in the transgenic animal unit. RAG2−/− and C3 transgenic mice, which selectively express the bacterial toxin C3-transferase under the control of CD2 promoter and locus control region in the thymus, have been described in detail elsewhere [10,18].
2.2
Flow cytometric analysis
Fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and biotin-conjugated antibodies were obtained from Pharmingen (San Diego, CA). Tricolour (PE/Cy5 and APC/Alexa750) conjugated antibodies and fluorophore-streptavidin conjugates were from Caltag (Burlingame, CA). Thymocytes were stained for cell surface markers and analysed on a FACS Calibur (Becton Dickinson Immunocytometry Systems Franklin Lakes CA). Data were analysed using CellQuest BDIS or FlowJo (Treestar Inc, Ashland OR) software. CD4−CD8− double negative thymocyte subsets were analysed for CD44 and CD25 expression following lineage exclusion of mature DP and SP cells as well as non-T cell lineage cells using a cocktail of biotinylated antibodies (CD4, CD8, CD3 B220, Mac-1, NK, Gr-1, and γδ) revealed with streptavidin-tricolour and costained with CD25-FITC, CD44-PE and Thy1.2-APC. Intracellular phospho-S6 and TCR-β intracellular staining was carried out on thymocytes pre stained to identify DN3 and DN4 subpopulations. Cells were subsequently fixed in 1% paraformaldehyde for 10 min at room temperature, washed in PBS and permeabilized with saponin buffer (0.5% saponin, 5% FBS, 10 mM HEPES [pH 7.4] in PBS) for 10 min at room temperature. Permeabilized cells were incubated with PE-conjugated TCRβ antibody and phospho S6 antibody for 45 min at room temperature, washed in saponin buffer and subsequently stained with FITC conjugated donkey anti rabbit IgG (Jackson Immunoresearch, West Grove PA). Cells were analysed on an LSR Flow Cytometer (BDIS).
2.3
RNA extraction
Thymocytes were isolated from 4- to 6-week-old RAG2−/−, CD2–C3 and C57BL/6 control mice. DN3 pre-T cells were purified firstly by removing CD4, CD8 DP cells using MACS CD4+ and -MACS CD8+ T cell isolation kits (Miltenyi Biotec, Ltd.) followed by magnetic autoMACS separation. CD4/CD8 depleted thymocytes were then labelled with Thy1.2-APC, PE conjugated CD4, CD8, CD44 and TCRγδ antibodies, FITC conjugated CD25 antibodies and the DN3 subpopulation (Thy1+CD4−/CD8−/CD44−/CD25+) was purified by cell sorting on a FACS Vantage (BDIS) using Cell Quest software (BDIS). All sorted cells were analysed by flow cytometry and only used for experiments if the purity was 95–98%. Total RNA was extracted from sorted cells using the Qiagen RNeasy kit according to manufacturers instructions. Total RNA was quantified using the RNA 6000 Nano LabChip® kit and analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA from up to 10 thymi were pooled for microarray analysis and quantitative PCR.
2.4
Microarray
Microarray analysis was carried out by the Finnish DNA Microarray Centre at the Centre for Biotechnology, Turku, Finland. For Affymetrix sample preparation, 100–1000 ng of total RNA was used as the starting material to synthesise target cRNA using the GeneChip® Eukaryotic Small Sample Target Labeling Assay Version II according to manufacturers’ instructions (Affymetrix, Santa Clara, CA). The cRNA target sample was hybridised to the Affymetrix Mouse Genome 430 2.0 Array (45,101 probes sets and expressed sequence tag (EST) clusters). Data was analysed with Affymetrix Gene Chip Operating Software (GCOS) version 1.1 and filtered according to recommendations of the manufacturer. Comparison analysis was used to compare the expression profiles from two arrays – results were obtained by analyzing the experimental array in comparison to the baseline array. The global method of scaling was used for normalizing hybridization intensity between arrays. The statistical algorithm used by GCOS defined the expression status of each gene-specific probe set as not changed, increased, marginally increased, decreased or marginally decreased. Genes that were absent or unchanged between comparisons were excluded from the results. Comparison analysis defines a gene as up-regulated if the signal log ratio between the baseline sample and the experimental sample is larger than 1 (2-fold) and the experimental sample is present. Similarly changed genes were further analyzed/classified using the gene ontology tool (www.Affymetrix.com) and represented by biological function. Gene lists were complied and sorted by genes that were either commonly or uniquely regulated in CD2-C3 and RAG2−/− thymocytes.
2.5
Real-time RT-PCR analysis
Purified total RNA (200 ng) was reverse transcribed using the iScript™ cDNA synthesis kit (BioRad). Real time RT-PCR was performed in a 96-well plate using iQ™ SYBR Green based detection (BioRad) on a BioRad iCycler in 20 μl reaction volume containing 1 μl cDNA (20 ng), 0.8 μl 10μM sense and antisense primers, 10 μl iQ™ SYBR Green supermix, and 4 μl nuclease free water. Each reaction was performed in duplicate and each experiment repeated in triplicate. 18S rRNA levels were used to normalise RNA concentrations between samples and the relative mRNA levels were calculated using the equation:where E is the efficiency of PCR, ct is the threshold cycle, u is the mRNA of interest, r is the reference gene (18S rRNA), s is the sample and c is the control sample. Primers used for RT-PCR were designed using Beacon Designer 2 software. Primer sequences are: Egr1 forward 5′-ACAGAAGGACAAGAAAGCAGAC-3, reverse 5-CCAGGAGAGGAGTAGGAAGTG-3′, Fos forward 5′-CTACTGTGTTCCTGGCAATAGC-3′, reverse 5′-AACATTGACGCTGAAGGACTAC-3′, Egr3 forward 5′-TGACCAACGAGAAGCCCAATC-3′, reverse 5′-GCTAATGATGTTGTCCTGGCAC-3′, Jun forward 5′-CGCCTCGTTCCTCCAGTC-3′ reverse 5′-ACGTGAGAAGGTCCGAGTTC-3′, JunB forward 5′-CTTCTACGACGATGCCCTCAAC-3′, reverse 5′-GTTCAAGGTCATGCTCTGTTTTAGG-3′, Nur77 forward 5′-CCTGTTGCTAGAGTCTGCCTTC-3′, reverse 5′-CAATCCAACACCAAAGCCACG-3′, 18S RNA forward 5′-GTAACCCGTTGAACCCCATT-3′, reverse 5′-CCATCCAATCGTAGTA-3′.
3
Results and discussion
Mice expressing the RhoA inhibitor Clostridium Botulinum C3 transferase under the control of the CD2 locus control region (LCR) have been described previously [18]. Loss of RhoA function in CD2-C3 mice results in loss of CD4/CD8 double positive thymocytes (Fig. 1A) with a block in thymocyte development at the DN3 stage (Fig. 1B). Rag2−/− mice also block thymus development at the DN3 stage (Figs. 1A and B) due to failed expression of the pre-TCR complex. Despite this superficial similarity, loss of Rho function does not prevent TCR beta locus rearrangements although there is a reduced frequency of DN3 cells expressing intracellular TCR-β subunits (Fig. 1C). Ectopic expression of transgenic TCR complexes cannot reverse the developmental block in CD2–C3 thymocytes demonstrating that failed expression of TCR complexes does not explain the developmental defects caused by loss of Rho function [18]. Rho thus appears necessary for pre-TCR function although these results do not discriminate between a direct role for RhoA in pre-TCR signalling or a role for this GTPase in the cytokine/stromal signalling pathways that synergise with pre-TCR signals to control pre-T cell differentiation [25–27].
CD2–C3 transferase mice provide a good model system to probe the immediate transcriptional consequences of losing RhoA function in pre-T cells since the CD2-LCR initiates expression of transgenes in T cell progenitors as they transit from the DN2 to DN3 stage of thymocyte development [18]. Accordingly, it is possible to isolate DN3 thymocytes ex vivo that have only just switched on expression of C3 transferase and prepare RNA for DNA microarray analysis. The Affymetrix Mouse Genome 430 2.0 array, representing 39 000 murine gene transcripts, was used to transcriptionally profile DN3 pre-T cells purified from the thymi of CD3–C3 transgenic mice. In these experiments DN3 thymocytes from wild type control mice were transcriptionally profiled and used for comparisons.
The microarray data were analyzed with the Affymetrix Gene Chip Operating Software (GCOS, version 1.1) by comparing the gene expression profiles of CD2–C3 pre-T cells to the profile of normal cells to reveal changes above or below wild type levels. DN3 pre-T cells expressed approximately 18 000 genes and there were only small changes in the transcriptional profile of CD2–C3 DN3 thymocytes compared to control wild type DN3s. We focused our analysis on ⩾2-fold changes, which were further analyzed using the Affymetrix gene ontology tool. A full list of the genes regulated 2-fold or greater by loss of RhoA function in DN3 thymocytes is shown in Supplementary Fig. 1. There were approximately 18 000 genes expressed in wild type DN3 thymocytes: loss of RhoA function caused a ⩾2-fold decrease in expression of 383 genes and a ⩾2-fold increase in expression of 190 genes. RhoA regulated genes in DN3 thymocytes encode proteins with diverse biological functions including serine/threonine kinases, protein phosphatases, enzymes that regulate protein biosynthesis.
This point is illustrated in Fig. 2A, which shows the genes whose expression is regulated more than 10-fold. The largest decrease was in expression of the gene encoding FosL2, a Fos family transcription factor (630-fold decrease) but other large changes included a subunit of eukaryotic translation initiation factor 2, Protein Kinase C beta and the membrane receptor CD69. There was also a loss of expression of genes encoding proteins that regulate nuclear structure and function such as genes encoding histones that are involved in nucleosome assembly. In a similar category there was reduced expression of mRNA encoding special AT-rich sequence binding protein 1 (SATB1) in CD2-C3 DN3s (Supplementary Fig. 1). SATB1 acts as a scaffold for chromatin modifiers and is known to regulate higher order chromatin structure and to regulate gene expression by acting as a “docking site” for several chromatin remodeling enzymes [28–30].
Of 55 genes whose expression was decreased ⩾5-fold, 23 encoded transcription factors; of 384 genes decreased ⩾2-fold, 63 were transcription factors (Fig. 2B). Transcription factors downregulated in CD2-C3 DN3 thymocytes included Kruppel family transcription factors KLF4, KLF6 and KLF9. There was also loss of expression of members of the immediate early gene family Egr1 and Egr3, AP-1 family members Fos, FosL2, FosB, Jun, JunB and downregulated expression of genes encoding Ets1 and Nurr77. To validate the microarray analysis, quantitative real-time PCR was used to compare expression of a selection of these different transcription factors in wild type versus CD2-C3 thymocytes (Fig. 2C). The experiments confirmed the reduction in Fos, Jun, JunB, Egr1 and Egr3 and NURR77 in CD2–C3 DN3s compared to wild type controls.
A number of gene targets for Egr transcription factors have been described [31] and in this context, RhoA inactivation caused loss of expression of multiple members of the MAP kinase phosphatase family DUSP 1, 2, 4, 6, 16. The latter are induced in negative feedback mechanisms that modulate MAP kinase activity and have been described as targets for Egr family transcription factors [31–33]. The loss of DUSP expression is thus almost certainly a secondary consequence of failed expression of Egr molecules. Egr1 and Egr3 are well characterised pre-TCR induced genes [34–38]. To verify this we compared the transcriptional profile of CD2-C3 DN3s with Recombinase gene 2−/− (RAG2−/−) DN3s which lack expression of the pre-TCR complex because they fail to rearrange their TCR beta locus. Of the 23 transcription factors downregulated ⩾5-fold in CD2–C3 thymocytes 18 were also downregulated in Rag2−/− DN3s (Fig. 2D) and these included Egr1, Egr3, Fos, Fosl2, FosB, Ets1, Nurr77. These microarray analyses were confirmed by quantitative real-time PCR analysis of wild type versus Rag2−/− thymocytes (data not shown). The common loss of expression of members of the Egr family in CD2–C3 and Rag2−/− pre-T cells is thus consistent with a RhoA requirement for the pre-TCR induced gene transcription. However, comparisons of the total number of genes regulated uniquely in CD2–C3 and Rag2−/− DN3s (Fig. 3) (VENN diagrams) indicate that there are a number of gene changes in CD2–C3 cells that are not seen in Rag2−/− DN3s. The full list of genes that were commonly and uniquely regulated 2-fold and greater in CD2 C3 versus Rag2−/− pre-T cells is shown in Supplementary Fig. 2. In terms of ⩾ 2-fold, gene repression, CD2-C3 and Rag2−/− DN3s had 180 gene targets in common with 202 genes being downregulated uniquely in CD2–C3 pre-T cells and 120 genes downregulated uniquely in Rag2−/− cells. In terms of ⩾2-fold gene increases, CD2–C3 and Rag2−/− DN3s had 65 gene targets in common, 129 genes that were increased uniquely in CD2-C3 DN3s and 182 increased uniquely in Rag2−/− cells (Fig. 3).
The common transcriptional changes caused by loss of the pre-TCR in Rag2−/− DN3s or loss of Rho function in CD2–C3 mice indicates that Rho function is necessary for some pre-TCR induced transcriptional responses. However, there are genes downregulated in DN3s lacking RhoA function that are unchanged in Rag2−/− DN3s (Supplementary Fig. 3) indicating that RhoA is not just required to mediate the preTCR induced gene program but may also participate in cytokine/stromal cell-initiated signalling pathways that control pre-T cell differentiation. The genes uniquely lost in CD3–C3 DN3 thymocytes include serine kinases (Protein Kinase C beta and Map3k14); transcription factors (SpiB and Dlx1) and regulators of protein biosynthesis (Eif2s3y) (Supplementary Fig. 3). One gene of interest lost in CD2–C3 DN3s but not Rag2 null DN3s was Hes-1 which is induced by Notch receptor-ligand interactions. Notch signalling is required throughout DN to DP stage of thymocyte development to support T cell metabolism and survival [39–42]. A RhoA requirement for Notch signalling has not been described but it is known that RhoA is necessary for integrin mediated cell adhesion and thymocyte cell migration [19]. Accordingly, decreased Hes-1 expression could reflect that pre-T cells lacking Rho function cannot make normal contacts with stromal cells that express the Notch ligands [43,42,44,45].
There were also unique transcriptional changes seen in Rag2−/− cells that were not found in the CD2–C3 DN3s (Supplementary Fig. 4) which indicates that the pre-TCR can regulate a program of gene transcription via RhoA independent pathways The unique changes in Rag2−/− cells not surprisingly included loss of expression of rearranged TCR-β subunits. One other gene downregulated in Rag2−/− DN3s but not CD2–C3 DN3s was RPS6, which encodes the ribosomal S6 subunit. In this respect, we recently described high basal levels of S6 phosphorylation in ex vivo β selected DN3s and no detectable S6 phosphorylation in ex vivo Rag2−/− DN3s [13]. The data in Fig. 4 quantify S6 phosphorylation in CD2–C3 DN3s compared to wild type cells. Normal DN3s are heterogeneous for phosphoS6 with the majority of cells being phosphoS6low but a significant percentage of cells are phosphoS6high. The DN3 thymocyte subpopulation can be subdivided into cells that have not yet completed TCR-β locus rearrangements and those that express a functional TCR-β subunit that allows surface expression and signalling of the pre-TCR complex. Analysis of TCRβ expression by intracellular staining revealed that DN3s that express icTCR-β chains are generally phosphoS6high whereas DN3s that are icTCR-β null are uniformly phosphoS6low. In CD2–C3 DN3s there were also high levels of phosphoS6 in TCR-β high cells. The presence of normal S6 phosphorylation in β selected DN3s lacking Rho function is a further indication that Rho is necessary for a subset of responses in DN3 thymocytes but does not globally block signalling.
One important question is whether the transcriptional changes in CD2–C3 DN3s explain why loss of RhoA function results in failed thymocyte differentiation? In this respect, microarray analysis of Rag2−/− pre-T cells revealed decreased expression of TCR-β subunits as a major defect (Supplementary Fig. 4) and failed expression of the TCR-β subunit is indeed responsible for the DN3 developmental block in Rag2−/− mice. In CD2–C3 DN3s there were several gene defects that would explain why RhoA function is essential for pre-T cell development. For example, RhoA is required for expression of Egr3 and Ets1 and loss of either of these transcription factors inhibits pre-T cell proliferation and is suboptimal for transition through early pre-TCR-dependent stages of thymocyte development [46,36]. Loss of individual Egr family or AP-1 transcription factors does not completely abrogate thymocyte differentiation but this reflects that there is considerable redundancy in AP-1 complexes due to the ability of different family members to pair as dimers [47–49]. The simultaneous elimination of Egr1, Egr3, Ets1 and multiple Fos/Jun family members in CD2–C3 DN3s would circumvent the possibility of redundancy [50,51]. This comprehensive loss of AP-1 activity in pre-T cell lacking RhoA function plus the decreased expression of other transcription factors would rapidly cause global changes in the transcriptional program of a cell. Moreover, the transcription factor defects would be exacerbated by the decreased expression of chromatin modifying enzymes such as SATB1.
In summary, loss of Rho function or failed expression of the pre-TCR complex in Rag2−/− null mice block T cell development at a common stage. The present data show that the genetic consequences of loss of Rho function versus loss of pre-TCR expression for T cell progenitors are not identical. The present results also show that loss of Rho function in pre-T cells results in downregulation of genes encoding members of the Fos/Jun and Early growth response (Egr) family of transcription factors. The collective loss of these transcription factors and the resultant secondary genetic changes explains why loss of RhoA function prevents pre-T cell development.


Dorsal hippocampal N-methyl-d-aspartate receptors underlie spatial working memory performance during non-matching to place testing on the T-maze

Previous lesion studies have suggested a functional dissociation along the septotemporal axis of the hippocampus. Whereas the dorsal hippocampus has been implicated in spatial memory processes, the ventral hippocampus may play a role in anxiety. However, these lesion studies are potentially confounded by demyelination of fibres passing through the lesion site, and the possibility of secondary, downstream changes in associated brain structures as a consequence of their chronic denervation following the lesion. In the present study, we have used the microinfusion of muscimol to temporarily inactivate either the dorsal or ventral hippocampus in order to re-examine the contribution of the hippocampal sub-regions to spatial memory. Microinfusion studies spare fibres of passage and offer fewer opportunities for compensatory changes because the effects are transient and short-lasting. Rats were infused prior to spatial working memory testing on a non-matching to place T-maze alternation task. Spatial working memory was impaired by dorsal but not ventral hippocampal inactivation. In a second experiment, infusion of the NMDAR antagonist, D-AP5, into dorsal hippocampus also impaired spatial working memory performance, suggesting that NMDAR function within the dorsal hippocampus makes an essential contribution to this aspect of hippocampal information processing.
1
Introduction
A role for the hippocampus in certain kinds of memory is well established. In rodents, the hippocampus has been particularly associated with spatial learning [1]. Complete hippocampal lesions have repeatedly been shown to produce robust and lasting impairments on spatial memory tasks [2,3]. More recently, however, these spatial memory functions have been ascribed specifically to the dorsal (or septal) portion of the rat hippocampus [4,5]. For example, selective cytotoxic lesions restricted to dorsal hippocampus produce spatial learning impairments on tasks like the Morris watermaze [6, 7, see also 8]. In contrast, lesions of the ventral hippocampus are without effect on spatial memory tasks, although conversely, they, but not dorsal hippocampal lesions, are associated with reduced anxiety [9–11]. This double dissociation suggests that different hippocampal sub-regions may mediate different aspects of hippocampal function [5].
Thus far, the conclusion that spatial learning is primarily a function of dorsal hippocampus has largely been based on the results of such cytotoxic lesion studies. However, this approach is potentially confounded in that any behavioural sequelae may be the result of indirect effects of the lesion procedure on other brain areas, rather than as a direct consequence of cell loss in the target region. For example, although the cytotoxic lesion approach is a clear improvement over more traditional techniques (e.g. aspiration, electrolytic or radiofrequency-generated lesions), in terms of sparing fibres of passage [12], there nevertheless remains some evidence of demyelination of nerve fibres passing through the lesion site even with this approach [13,14]. Furthermore, it is also possible that any behavioural effects of the lesion could reflect a secondary, downstream change in an associated brain structure as a consequence of the chronic denervation of that structure following the lesion [15]. It is therefore important to re-examine the dorsal/ventral dissociation using alternative experimental approaches. For example, it is possible to produce a temporary, reversible inactivation of a specific brain region by microinfusing the GABAA agonist muscimol [16], which avoids many of the potential confounds associated with the lesion approach. Microinfusion studies offer fewer opportunities than lesion studies for compensatory changes because the effects of the manipulation are transient and short-lasting. They also permit within-subjects comparisons. Somewhat surprisingly, the effects of dorsal and ventral hippocampal inactivation on spatial working memory performance have never been fully assessed.
In the present study, we therefore compared the effects of dorsal and ventral hippocampal infusions of muscimol on spatial learning using a spatial working memory, non-matching to position (discrete trial, rewarded alternation) paradigm on the elevated T-maze [17]. This task is especially sensitive to hippocampal dysfunction (see effect sizes in [7], p. 1184), and is dramatically impaired by dorsal but not ventral hippocampal lesions [7,9,18]. We therefore compared dose-response functions for muscimol infusions into both hippocampal sub-regions (Experiment 1).
In a second experiment, the importance of N-methyl-d-aspartate receptor (NMDAR) function, specifically in the dorsal hippocampal subregion, for spatial working memory performance was assessed. The role of NMDARs in spatial working memory is well established [19,20]. Intracerebroventricular (i.c.v.) infusion of the NMDAR antagonist, d(−)-2-amino-5-phosphonopentanoic acid (AP5) resulted in a delay-related impairment in choice accuracy on the same spatial, non-matching to place T-maze task [21]. However, the i.c.v. route of drug delivery results in NMDAR blockade in numerous brain regions, and not just the hippocampus [22]. In view of the proposal that it is the dorsal subregion of the hippocampus that is crucial for spatial working memory performance, we therefore also examined the effects on spatial non-matching to position of direct infusions of AP5 specifically into dorsal hippocampus (Experiment 2).
2
Methods
2.1
Subjects
Thirty male Lister-hooded rats (Harlan Olac, Bicester, UK; 250–363 g at the time of surgery) took part in Experiment 1. Nine male Lister-hooded rats (270–370 g at the time of surgery) took part in Experiment 2. All rats were experimentally naïve and maintained on a 12 h light/dark cycle (lights on at 7:00 a.m., with testing during the light phase). During the experiment, the animals were maintained at ∼85% of their free-feeding weight but had access to water ad libitum. Rats were housed in groups of 2–3 prior to surgery but were singly housed after cannulae implantation. The experiments were conducted in accordance with the United Kingdom Animals Scientific Procedures Act (1986); under project license number PPL 30/1505.
2.2
Apparatus
In Experiment 1, rewarded alternation took place on an elevated (∼1 m above the floor), low-walled wooden T-maze consisting of a start arm (80 cm long; 10 cm wide) joined to two identical goal arms (60 cm × 10 cm), with each arm surrounded by a 1 cm ridge. In Experiment 2, rewarded alternation took place on an elevated Y-maze (∼80 cm above the floor). Each of the three arms (50 cm × 9 cm) was surrounded by a 0.5 cm ridge and extended from a hexagonal central platform (14 cm diameter). The T- and Y-mazes contained stainless-steel food wells at the far end of each goal arm and each maze was located in a well-lit testing room containing prominent extra-maze cues such as wall posters.
2.3
Procedure
In both experiments, rats were trained pre-operatively. Habituation to the maze took place over a five-day period. For the first three days rats were placed on the maze in pairs and left to explore and collect food rewards for 10 min. During days four and five, the rats were placed individually on the maze and allowed to explore freely for five minutes. By this stage all the rats ate from the food wells at the ends of the arms. Training on the rewarded alternation task then followed.
Each trial in rewarded alternation had two runs: in the first, one of the goal arms was blocked, allowing the rat to enter only the other goal arm, whereupon it received 1 food pellet (45 mg Rodent Diet Formula A/I, Noyes, Lancaster, NH). During the second run, the block was removed and the rat was placed on the maze and given a free choice of either arm. Rats received two pellets for choosing the previously unvisited arm (i.e. for alternating). Choosing the arm previously visited in the sample run yielded no reward. The time between the sample and the choice runs was approximately 10 s (Experiments 1 and 2a) or 30 s (Experiment 2b). Left/right allocations for the sample and choice runs were pseudo-randomised over ten trials per day, with no more than three consecutive sample runs to the same side. The inter-trial interval was ∼3–4 min. Prior to surgery, each rat was trained on the task until they achieved a criterion of at least 80% correct alternation over two consecutive days.
In Experiment 1, allocation to surgery groups (dorsal or ventral cannulation) was based on pre-operative performance. Rats were anaesthetised with Avertin (0.29 g/kg; i.p.) and placed in a stereotaxic frame with the head level between bregma and lambda. An incision of the scalp was made along the midline, followed by a craniotomy. Stainless-steel guide cannulae (23-gauge, 12 mm) were implanted bilaterally into either the dorsal (n = 14; AP: −3.6 (from bregma); M–L: ±2.8 (from bregma); D–V: −0.8 (from brain surface)) or ventral HPC (n = 16; AP: −5.2 (from bregma); M–L: ±4.9 (from bregma); D–V: −3.6 (from brain surface)). In Experiment 2, cannulae were only implanted into the dorsal HPC (n = 9; coordinates as above). Cannulae were secured with three skull screws and acrylic dental cement. Microinjectors (31-gauge, 14 mm) were 2 mm longer than the guide cannulae giving a targeted D–V coordinate (from brain surface) of either −2.8 mm (dorsal) or −5.6 mm (ventral). Stylets were inserted into the guide cannulae to prevent infection and blockages.
Rats were allowed a minimum of five days post-surgical recovery after which they received training in the absence of any infusions. Rats failing to score 80% correct alternation on these sessions were given additional training.
2.4
Drugs
In Experiment 1, muscimol (C4H6N2O2(1/2 H2O); Tocris, Bristol, UK) was initially dissolved in normal saline (0.9% NaCl) at a concentration of 1 mg/ml. This stock was then diluted to create individual aliquots at each of four concentrations (0.6 mg/ml, 0.3 mg/ml, 0.15 mg/ml, and 0.075 mg/ml), which were then frozen. Saline served as the vehicle. In Experiment 2, muscimol (0.3 mg/ml) and AP5 (d(−)-2-amino-5-phosphonopentanoic acid; 5.9 mg/ml) were dissolved in PBS (pH 7.4), which served as vehicle.
On infusion days, the aliquots were defrosted and used to back-fill the microinjectors, which were connected via polyethylene tubing to a pair of Hamilton syringes (10 μl) driven by a microsyringe pump (SP250i, World Precision Instruments, England). Each rat was restrained firmly in a towel, the stylets were removed, and the microinjectors inserted through the guide cannulae into the hippocampus. Drug or vehicle (0.5 μl per side) was infused into the dorsal or ventral hippocampus at a rate of 1 μl/min (i.e. for 30 s), and the microinjectors were kept in place for an additional 60 s to allow the drug to diffuse. Infusions into each hemisphere were made simultaneously and were given 15 min prior to testing. In Experiment 1, rats received five infusions in total (saline and four doses of muscimol: 0.3 μg/side, 0.15 μg/side, 0.075 μg/side, 0.0375 μg/side). In Experiment 2a, rats received three infusions (PBS, AP5: 2.95 μg/side, muscimol: 0.15 μg/side) and in Experiment 2b, the same rats received a further 4 infusions (PBS × 2, AP5: 2.95 μg/side × 2), making seven infusions in total. Infusions were given in a counterbalanced, pseudo-random order with at least 48 h between each infusion. In addition, performance was assessed again 24 h after each infusion and rats failing to score 80% or more on these drug-free sessions received extra training before the next infusion was administered.
2.5
Histology
On completion of the experiment, rats were perfused transcardially and their brains were removed, sectioned, and stained with cresyl violet for verification of the injection sites.
3
Results
3.1
Experiment 1
In Experiment 1, of the 30 rats that began the experiment, 23 rats completed all testing and had accurate bilateral cannulae placements and were included in the final analyses (10 dorsal, 13 ventral; Fig. 1). The intra-hippocampal implantation of guide cannulae did not result in any post-surgical deficits in rewarded alternation in either dorsal (mean score = 94%) or ventral (mean score = 90%) cannulated rats. Indeed post-operative performance was slightly better than pre-operative performance in both groups. Student's t-tests carried out for each group confirmed that there were no differences between pre-operative and (uninfused) post-operative performance (dorsal: t(9) = −0.483; p > 0.2; ventral: t(12) = −1.15; p > 0.2).
The amount of alternation following saline infusion into either dorsal or ventral hippocampus was similar and comparable to uninfused performance (dorsal = 88%; ventral = 85%). In contrast, bilateral infusion of muscimol into the dorsal hippocampus produced a decrease in alternation levels (see Fig. 2). These data were analyzed using a repeated-measures ANOVA [model: cannula placement2 × (drug treatment5 × S23)]. The ANOVA revealed a significant effect of cannula placement (F1,21 = 14.44; p < 0.001) and drug treatment (F4,84 = 9.01; p < 0.001) but the interaction was not quite significant (F4,84 = 2.05; p = 0.09).
Analysis of simple main effects and subsequent pairwise comparisons (using Sidak's method, see [23]) found an effect of cannula placement at three doses of muscimol: 0.0375 μg/side (F1,21 = 8.94; p < 0.01), 0.075 μg/side (F1,21 = 6.94; p < 0.05) and 0.3 μg/side (F1,21 = 4.45; p < 0.05) with the dorsal group exhibiting greater impairment than the ventral group. The groups did not differ following saline infusion (p > 0.2) or when the muscimol dose was 0.15 μg/side (p > 0.1). Simple main effects analysis also revealed an effect of drug within the dorsal cannula placement group (F4,18 = 6.86; p < 0.005), with all doses of muscimol leading to performance impairment compared to saline infusion. In contrast, there was no effect of drug within the ventral placement group (F4,18 = 2.7; NS). In other words, although there was some suggestion that performance was decreasing as the dose of muscimol increased (see Fig. 2), no individual dose of muscimol into the ventral hippocampus significantly impaired performance compared to saline infusion.
On alternate days, the rats received 10 trials of rewarded alternation in the absence of any infusions to investigate the possibility of permanent or progressive damage to the targeted regions. Importantly, this data showed that rats from both groups alternated on more than 85% of trials 24 h after receiving infusions. Post-infusion performance of dorsal and ventral cannulated rats was investigated with a repeated-measures ANOVA [model: cannula placement2 × (block5 × S23)]. The ANOVA found no effect of cannula placement (F1,21 = 2.37; NS), or block (F4,84 = 0.49; NS), and there was no interaction (F4,84 = 1.99; NS). This result demonstrates that the T-maze impairments identified in the previous analysis were reversible and only evident while the drug was actually present in the hippocampus.
3.2
Experiment 2
In Experiment 2, all nine rats had accurate bilateral dorsal hippocampal cannulae placements (Fig. 1). Experiment 2 was divided into two stages. Procedurally, Experiment 2a was identical to Experiment 1. Experiment 2b was identical to Experiment 2a except that a 30 s delay was interposed between the sample and choice runs of each trial of rewarded alternation (see [21]).
In Experiment 2a, the amount of alternation following PBS infusion into the dorsal hippocampus was similar and comparable to uninfused performance (uninfused = 90%, PBS = 83%). In contrast, there was a substantial drop in choice accuracy following the infusion of either muscimol (58%) or AP5 (67%). A one-way repeated-measures ANOVA [model: drug condition2 × S9], found a main effect of drug condition (F2,16 = 6.3; p < 0.01) and post hoc pairwise comparisons (Student's Newman–Keuls) revealed significant differences between the vehicle and both drug conditions (p < 0.05) but not between the two drug conditions (p > 0.2).
The introduction of the 30 s delay in Experiment 2b did not lead to a further drop of performance in either infusion condition (PBS = 83%; AP5 = 72%). Although not strictly appropriate (since the data were not derived in a counterbalanced manner), a repeated measures ANOVA was performed on the combined data of Experiments 2a and 2b (see Fig. 3). The ANOVA revealed a main effect of drug condition (F1,7 = 11.75; p < 0.05) but no effect of delay or drug condition × delay interaction (both F < 1; NS).
Finally, performance accuracy (90%) during drug-free testing carried out after the last infusion confirmed that there was no lasting damage due to cannulae implantation or the microinjection process.
4
Discussion
Experiment 1 showed that selective inactivation of the dorsal hippocampus results in significant impairment of spatial working memory performance whereas equivalent inactivation of ventral hippocampus does not. This result is consistent with previous lesions studies [7,18], but advances on these earlier findings by demonstrating that such dorsal/ventral differences are not due to indirect effects such as demyelination or compensatory changes that result from permanent brain lesions. Also, the impairment resulting from infusion of AP5 directly into the dorsal subregion (Experiment 2) identifies the importance of N-methyl-d-aspartate receptor (NMDAR) function in the dorsal hippocampus for spatial working memory performance.
This data, and that obtained from previous cytotoxic lesions studies, are consistent with findings from a variety of different approaches which suggest a functional specialization along the septotemporal (dorsal/ventral) axis of the hippocampus. Evidence from electrophysiological single unit recording studies in both rats [24,25] and primates [26], from c-fos activation studies [27], and from structural magnetic resonance imaging studies [28], are all consistent with a functional dissociation between dorsal (posterior in primates) and ventral (anterior in primates) hippocampus, and with a preferential role for dorsal hippocampus in spatial learning and memory.
To our knowledge, this experiment constitutes the first demonstration of differential effects of dorsal and ventral hippocampal inactivation on spatial working memory performance using T-maze rewarded alternation. It is worth noting, however, that the effects of selective inactivation of dorsal and ventral hippocampus on delayed alternation tasks have been examined previously using operant paradigms, but with mixed results. For example, Mao and Robinson [29] trained rats to press a lever on the left or right-hand side of the front wall of the operant chamber as indicated by the presence of a light stimulus (equivalent to the sample phase in the present study), then press a lever on the rear wall before being given a free choice of either front wall lever (equivalent to the choice phase in our study). The rats were required to alternate lever presses on the front wall in order to obtain food rewards. Although this task has some similarity to our T-maze task (both require alternation behaviour), bilateral infusions of muscimol into the dorsal hippocampus did not affect the proportion of correct responses. It is worth pointing out, however, that the doses used by Mao and Robinson were lower than the doses used in the present study (0.003–0.01 μg compared to 0.0375–0.3 μg in the present study). It is also worth noting that in their study dorsal infusions of higher doses of muscimol (0.03–0.2 μg) interfered with the rats’ ability to perform various non-spatial aspects of task performance such as correct lever presses at the sample stage and the rear lever press between the choice and sample phases. In a separate study, Maruki et al. [30], trained rats to alternate between pressing the left and right lever on each trial. To prevent mediating strategies, the rat was required to make a touch response to the central food-well during the ITI. Using this continuous alternation paradigm, the authors found that infusions of muscimol (0.07 μg/side) into the dorsal hippocampus reduced correct responding when the ITI was 20 s but not when it was 3 s. In a second experiment, they reported no effects of ventral infusions of muscimol relative to saline vehicle infusions on the same task. However, inspection of the data across the two experiments shows that the levels of performance in rats receiving muscimol infusions into dorsal hippocampus (70.9% correct) and in rats receiving muscimol into ventral hippocampus (72.3% correct) were virtually indistinguishable. The present study thus provides the first clear demonstration that spatial working memory performance is sensitive to dorsal but not ventral hippocampal inactivation.
As noted by Mao and Robinson [29] direct intra-hippocampal infusion carries the risk of non-specific effects on performance. For example, it is possible that unequal quantities of muscimol entering each hemisphere could lead to a turning bias. First, it is worth pointing out that in our study both hemispheres were infused simultaneously. Second, in Experiment 1, rats receiving dorsal HPC infusions exhibited side preferences (i.e. they exclusively entered the same goal arm on the choice phase of each trial of a session) in only 13 of the 40 sessions (10 rats × 4 muscimol doses). It is also worth pointing out that such side preferences are commonly observed in rats with permanent HPC lesions and in our experience these preferences are not dependent on the size of the lesion in one particular hemisphere. They are generally considered to be a consequence, rather than a cause, of the memory impairment. Furthermore, the fact that in Experiment 1, errors were made to both goal arms in almost 70% of sessions argues against a performance account based solely on side-preferences elicited by unequal quantities of muscimol entering each hemisphere.
An alternative account of the septotemporal differences found in Experiment 1 is that the spread of the drug within the hippocampus is simply greater when infused into dorsal than ventral HPC, effectively creating a larger dorsal than ventral HPC inactivation. Without autoradiography to determine the extent of muscimol diffusion we cannot rule out this possibility completely. However, when the data from the present study are considered along with the previous cytotoxic lesion studies, such an account seems unlikely. Evidence from permanent lesion studies suggests that lesion size itself is not the critical determinant of degree of dysfunction. For example, Moser and colleagues [6] found that rats with lesions encompassing 20–40% of HPC tissue starting at the ventral pole showed normal spatial learning in the watermaze, whereas rats with lesions of 20–40% of HPC tissue starting at the dorsal pole were profoundly impaired. Furthermore, we have previously tested rats with dorsal and ventral cytotoxic lesions of very similar sizes (50–55%) on the same T-maze rewarded alternation task as used in the present study, and have found that whereas dorsal lesioned animals resemble animals with complete hippocampal lesions, exhibiting chance levels of performance, ventral lesioned animals perform as well as sham operated controls [9]. These data strongly suggest that lesion location, and not lesion size, is the critical factor.
In the present study, although no dose of muscimol into the ventral HPC significantly impaired performance compared to vehicle, there was some suggestion of a dose related decline in performance (see Fig. 2). This could reflect a role, albeit more limited, for the ventral hippocampus in spatial information processing. However, it is also possible that this slight drop in performance may reflect the spread of muscimol into mid-hippocampal and/or dorsal regions. In this regard, it is worth re-iterating that ventral hippocampal lesions, removing approximately 50% of the structure starting from the temporal pole, had absolutely no effect on performance on this very same spatial working memory, rewarded alternation task [7,9] Therefore, the evidence suggests that spatial working memory tasks such as rewarded alternation on the T-maze, like spatial reference memory tasks such as the standard fixed location version of the watermaze, are sensitive to dorsal but not equivalently sized ventral HPC lesions or inactivations.
Nevertheless, it may be unwise to completely exclude a role for the ventral HPC in spatial information processing. Single-unit studies have shown that cells selective for specific spatial locations (“place cells”) do exist in ventral HPC, albeit they are fewer in number and their place fields are larger than those in dorsal HPC. Although the electrophysiological data are limited, McNaughton and colleagues have suggested a septotemporal gradient in spatial selectivity with higher resolution spatial discriminations processed in dorsal HPC [24,25]. In this way, cells in dorsal HPC might code for specific locations within a particular environment whereas ventral cells might code for different environments or contexts, although this may be just a subset of the contextual information that can be processed by the ventral subregion [5]. It remains to be seen if tasks requiring discrimination between different spatial environments or contexts are more sensitive to lesions of the ventral HPC.
The results of Experiment 2 show that NMDARs in dorsal hippocampus contribute to performance on spatial working memory tasks. Previous work in this laboratory showed that i.c.v. infusion of AP5 impaired performance on the same rewarded alternation task [21]. The present results now suggest that this effect is, at least in part, due to the blockade of NMDARs in dorsal hippocampus. Similar spatial working memory impairments have also been observed following AP5 infusions into dorsal hippocampus on a spatial, matching-to-position version of the watermaze task [19]. Both of these results are consistent with the hypothesis that NMDAR-mediated forms of synaptic plasticity, similar to experimentally induced long-term potentiation [LTP; 31, 32], may underlie a flexible, rapidly acquired form of spatial memory, as typified by these spatial working memory tasks [20,33].
The observation that introducing a further 20 s delay failed to exacerbate the spatial working memory deficit might be considered surprising. In contrast to the present results, Tonkiss and Rawlins [21] showed that increasing the delay by 20 s did in fact increase the impairment in rats infused with AP5 i.c.v., on the same T-maze spatial working memory task. However, it is worth pointing out that in both cases, the effect of increasing delay was not investigated as part of a fully counterbalanced design, and therefore some caution is required when interpreting these results. Steele and Morris [19] did report a delay-dependent impairment in their matching to position watermaze task with a fully counterbalanced design, although it is worth noting that performance at the shortest delay was not consistently spared in their study.
We have also recently tested genetically modified mice in which the NR1 subunit of the NMDAR has been selectively deleted, specifically from the dentate gyrus subfield of the hippocampus [34]. These mice exhibit deficits in LTP in both the medial and lateral aspects of the perforant path, whereas LTP in the CA1 subfield is normal. We assessed spatial memory on the radial maze in these mice, and found that they exhibited a very specific spatial working memory deficit. As with the present study, the animals displayed a spatial working memory deficit at the shortest-possible delays that we were able to test. It is of course possible that at even shorter delays performance may be spared in the AP5 rats and in the dentate gyrus NR1 knockout mice. Equally, it could be that by blocking NMDAR-dependent synaptic plasticity in the hippocampus, we have disrupted an existing spatial representation of the environment, or prevented the expression of memory in some way, for example, by impairing the ability to select the appropriate spatial response on the basis of information retrieved from memory (both of which might be expected to result in a delay-independent deficit). Against this, the dentate gyrus-specific NR1 knockout mice displayed perfectly normal acquisition and performance on the reference memory component of the radial maze task (i.e. learning which 3 arms were baited and which three arms were never baited). It may therefore be that disrupting hippocampal synaptic plasticity prevents the animals from rapidly forming a short-term spatial representation of the maze arm visited on the sample run. This ability to record or represent the recent experience of a particular spatial location or stimuli (i.e. the maze arm) is likely to be of crucial importance for efficient spatial working memory performance [35].
In conclusion, the present research has confirmed and extended previous findings from lesion studies, namely that the dorsal hippocampus has a greater involvement in spatial working memory than the ventral hippocampus. Furthermore, NMDAR activation within the dorsal hippocampus makes an essential contribution to this aspect of hippocampal information processing.

dorsal
hippocampus
spatial memory
ventral
muscimol
ap5


Balance training program is highly effective in improving functional status and reducing the risk of falls in elderly women with osteoporosis: a randomized controlled trial

Introduction The purpose of this study was to investigate the effect of a 12-month Balance Training Program on balance, mobility and falling frequency in women with osteoporosis.
Introduction
Osteoporosis is a debilitating, widespread disease, which affects approximately 55% of the population above 50 years old in the USA [1]. Falls among the elderly, especially for those with osteoporosis, are associated with high morbidity and mortality and can involve high-cost medical intervention [2]. In fact, falls are responsible for 90% of the growing increase in hip fractures [3] and are the sixth cause of death among patients aged over 65 [4].
Falls are multifactorial, and their causes are categorized as intrinsic (personal) and extrinsic (environmental) factors [5, 6]. Some examples of intrinsic factors include: altered balance, neurological diseases, sensory deterioration, musculoskeletal diseases, postural hypertension and the use of medication [7].
Research shows that altered balance is the greatest collaborator towards falls in the elderly [6, 8–12], with a high correlation between balance deficit and the incidence of falls [13, 14].
For this reason, studies regarding the risk of falling in osteoporosis are of high priority in clinical intervention. Diminishing the incidence of falls is a health priority, which reflects on both the quality of life and health costs [2].
Moreover, evidence suggests that exercise reduces the risk of fractures, showing an effect on the maintenance of bone mass and, more importantly, improving postural stability, mobility and, consequently, diminishing the risk of falls [15]. Indeed, improving balance should be an objective in the prevention of falls [16].
However, knowledge regarding balance deficit and the probability of falls is limited and controversial [17]. The literature shows that exercise may or may not be efficient in the control of falls [18], and the impact of prevention programs with balance training in diminishing falling frequency has yet to be established, principally in women with osteoporosis.
Balance training has been investigated in healthy elderly individuals [3, 16, 19–21]; however, only one study regarding balance training in women with osteoporosis exists. Since patients with osteoporosis are at greater risk of fractures resulting from falls, further research in this group should be prioritized [22].
The purpose of the present study was to investigate the effect of a 12-month balance training program on functional and static balance, mobility and falling frequency in women with osteoporosis.
Patients and methods
Patient sample
Sixty-six elderly women aged over 65 years old were consecutively selected from patients of the Osteometabolic Disease Outpatient Clinic of the Rheumatology Division, University of São Paulo. Only patients with osteoporosis, classified according to the World Health Organization [23] were included; with a bone mineral density (BMD) T-score lower than −2.5 standard deviation (SD), in the lumbar spine, femoral neck or total femur region.
The following women were excluded: those with secondary osteoporosis, visual deficiency, severe auditive deficiency, or vestibular alteration of important clinical status, such as women who used assisted walking devices or who were unable to walk independently more than 10 meters [24]; those who planned to be out of town for more than 4 weeks during the 12-month study; and women who presented absolute or relative contraindications for exercise training according to the American College of Sports Medicine [25].
The patients were randomized consecutively into two groups: the group submitted for the Balance Training Program (Intervention Group), consisting of 34 patients; and the Control group, consisting of 32 patients without intervention. The Control group only received treatment for osteoporosis and orientation to prevent falls and return regularly (3-monthly follow-ups) to the Osteometabolic Disease Outpatient Clinic. All patients read and signed a term of free informed consent that described the procedures which would be realized during the research.
Measured variables: interview and medical chart records
Personal, family and clinical data were evaluated through an interview and medical chart records, with emphasis on the history of fractures, number of falls in the preceding year, use of medication for osteoporosis, and use of medication that favored the risk of falling, such as hypnotics, hypotensors and antidepressants.
Functional state evaluation
Static and dynamic balance and mobility were evaluated in all patients, before and at the end of the trial, by a physiotherapist who was blinded to the distinct group (Intervention, Control).
Functional balance
The Berg Balance Scale (BBS) is based on 14 items common to daily life activities used to evaluate functional balance [26]. The maximum score that can be achieved is 56, and each item possesses an ordinal scale of five alternatives which varies from 0 to 4 points.
The test is simple, easy to administer and accompanies the evolution of elderly patients. It only requires a ruler and a watch and takes approximately 15 minutes to execute [26]. A score lower than or equal to 45 is considered evidence of altered balance [27].
Static balance
Static balance was evaluated by the Clinical Test of Sensory Interaction for Balance (CTSIB), which consists of six sensory conditions (1: eyes open and firm surface, 2: eyes closed and firm surface, 3: eyes open, visual conflict and firm surface, 4: eyes open and unstable surface, 5: eyes closed and unstable surface, and 6: eyes open, visual conflict and unstable surface).
Static balance is considered to be altered when an individual cannot remain at least 30 seconds in each of the six conditions [28].
Improvement in the test was defined as the capacity to complete the test during the final evaluation when unable to complete the same in the initial evaluation.
Functional mobility
Functional mobility was evaluated by the Timed “Up & Go” Test (TUGT) [29], which registers the time an individual takes to get up out of a chair, walk 3 meters, turn around, walk back and sit down again.
Elderly individuals without balance deficit are capable of completing the test in less than 10 seconds.
Falls
The number of falls in the year prior to the study [30] was solicited and noted in the initial evaluation and at the end of the trial (final evaluation). During the year of the study, patients in both groups received a diary and were orientated to write down the days that they fell.
At the end of the study, the difference in the number of falls/patient (final evaluation - initial evaluation) was compared between the Intervention Group and Control.
Intervention
The Balance Training Program consisted of 1 hour of exercises realized once a week, with a total of 40 classes, supervised by an experienced physiotherapist. This program was realized in a club (Associação Atlética Acadêmica Oswaldo Cruz - AAAOC) belonging to the Clinics Hospital, School of Medicine, University of São Paulo, located near to the Hospital. The balance exercises described by Tinetti and Suzuki [3, 11] were used. The type and mild to moderate intensity of the exercises used in the present study were chosen so that they could also be performed by elderly patients at home [3]. A list of weekly attendance controlled the absences of each patient.
Basic warm-up and stretching exercises
Prior to training, the patients participated in 15 min of warm-up and stretching exercises, consisting of head rotation, shoulder rotation and stretching of the upper and lower limbs. Walking was performed for 15 min with the supervision of a physiotherapist, who associated exercises for the upper limbs throughout the walk.
Balance training
Balance was realized in dynamic and static positions for a period of 30 min. This consisted of walking in the tandem position (one foot in front of the other), walking on the tips of the toes and on the heel, walking sideways, walking while raising the leg and the contra-lateral arm, standing on one leg, and standing in the tandem position, while gradually increasing the period of permanence in these last two static positions [3, 31].
Home-based exercises
The patients were instructed and encouraged to continue the same exercises at home at least three times a week for 30 min. A manual with instructions and illustrations for each exercise was distributed. The frequency of participation in the home-based exercises was noted each week by the physiotherapist.
Data analysis
Sixty-six consecutive patients were randomized in the present study. Data analysis was realized on 60 patients (30 Intervention Group and 30 Control), as six patients desisted (four Intervention Group and two Control). In the Intervention Group the reasons for desistance were: physical limitations as a result of falls (n = 1), foot pain (n = 1), personal reasons (n = 2); while in the Control group the reasons were: physical limitations as a result of falls (n = 1), personal reasons (n = 1).
The data were expressed as the mean and standard deviation (SD) for each variable and differences between the Intervention and Control groups were tested by the Student’s t-test or Mann-Whitney test. The Chi-square test or Fisher’s exact test was used to compare the number of patients in both groups (Intervention Group vs Control) for hypnotic and diuretic drug use, fracture history, CTSIB conditions (1–6) and improvement in CTSIB conditions. P values <0.05 were considered significant.
Results
The basal characteristics of the patients of both groups were similar in relation to age, body mass index (BMI), fracture history, osteoporosis treatment, diuretics and hypnotics/antidepressants use and bone mineral density (BMD), with no statistically significant differences between the two groups (Table 1).
Table 1Data at the onset of the study in relation to anthropometric parameters, fracture history, medication use and bone mineral density values (T-score) in the Intervention and Control groupsVariableIntervention (n = 30)Control (n = 30)p-valueAge, years74.57 ± 4.8273.40 ± 4.610.342*BMI, kg/m224.39 ± 4.4926.51 ± 5.320.100*Fracture history, n (%)13 (43.3)16 (53.3)0.438***Medication used for OP, n2.37 ± 1.502.30 ± 0.880.498**Hypnotics/Antidepressants, n (%)6 (20.0)7 (23.3)0.754***Diuretics, n (%)14 (46.7)16 (53.3)0.606***Lumbar spine, T-score−2.83 ± 1.07−2.62 ± 1.120.470**Femur neck, T-score−2.70 ± 0.75−2.75 ± 0.900.821**Total femur, T-score−2.10 ± 1.26−2.10 ± 1.090.990**Data expressed in means ± SD or percentageBMI: body mass index, OP: Osteoporosis*Student’s t-test**Mann-Whitney test***Chi-square test
Similarly, in the first evaluation, no differences occurred when comparing the Intervention Group and the Control Group in reference to: Berg Balance Scale (BBS) score, the number of patients that could not complete the Clinical Test of Sensory Interaction for Balance (CTSIB) in the six sensory conditions, the Timed “Up & Go” Test (TUGT), and the number of falls/patients in the preceding year (p > 0.05) (Table 2).
Table 2Data at the onset of the study for: Berg Balance Scale (BBS) score, number of patients that could not complete the Clinical Test of Sensory Interaction for Balance (CTSIB: condition 1: eyes open and firm surface; condition 2: eyes closed and firm surface; condition 3: eyes open, visual conflict and firm surface; condition 4: eyes open and unstable surface; condition 5: eyes closed and unstable surface; condition 6: eyes open, visual conflict and unstable surface), Timed “Up & Go” Test (TUGT), and number of falls/patient in the preceding year in Intervention Group and Control Intervention (n = 30)Control (n = 30)p valueBBS, score48.80 ± 4.1048.13 ± 5.360.900*CTSIB condition 1, n (%)0 (0.0)1 (3.3)1.000**CTSIB condition 2, n (%)2 (6.7)1 (3.3)1.000**CTSIB condition 3, n (%)2 (6.7)2 (6.7)1.000**CTSIB condition 4, n (%)4 (13.3)4 (13.3)1.000**CTSIB condition 5, n (%)15 (50.0)12 (40.0)0.604**CTSIB condition 6, n (%)12 (40.0)9 (30.0)0.589**TUGT, seconds14.31 ± 4.0313.86 ± 3.430.610*Falls/patient preceding year, n1.20 ± 1.880.87 ± 0.860.745*Data expressed in means ± SD or percentage.*Mann-Whitney test**Fisher’s exact test
Adherence rate
A high level of adherence was observed. Sixty percent of the patients participated in all of the exercise sessions at the club and absences occurred with the following justifications: doctor’s appointment, the realization of laboratory exams or for personal reasons. In relation to home-based exercise, 76.67% of the patients realized exercises at least once a week, 40% of the patients exercised every day and 36.67% from one to four times a week.
Comparison between the Intervention Group and Control
The difference in BBS score (final—initial evaluation) was greater in the group which suffered intervention (5.5 ± 5.67 vs − 0.5 ± 4.88, p < 0.001) (Table 3).
Table 3Differences (final evaluation—initial evaluation) in: Balance Berg Scale (BBS) score, number of patients showing improvement in Clinical Test of Sensory Interaction for Balance (CTSIB condition 5: eyes closed and unstable surface; condition 6: eyes open, visual conflict and unstable surface), time of Timed “Up & Go” Test (TUGT), and number of falls/patient in the Intervention Group and Control Intervention (n = 30)Control (n = 30)p-valueDifference BBS, score 5.5 ± 5.67−0.5 ± 4.88<0.001*CTSIB condition 5, n (%)13 (43.3)1 (3.3)<0.001**CTSIB condition 6, n (%)12 (40.0)1 (3.3)0.001**Difference TUGT, seconds−3.65 ± 3.61+2.27 ± 7.18<0.001*Difference of falls/patient, n−0.77 ± 1.76+0.03 ± 0.960.018*Data expressed in means ± SD or percentage.*Mann-Whitney test**Chi-square test
Similarly, the percentage of patients in the Intervention group whose static balance improved in two sensory conditions (CTSIB, condition 5: eyes closed, unstable surface; and condition 6: eyes open, visual conflict, unstable surface) was statistically significant when compared to Control (CTSIB condition 5: 13 patients vs 1, p  < 0.001; CTSIB condition 6: 12 patients vs 1, p < 0.001) (Table 3).
Equally, a significant difference in the functional mobility, as measured by the TUGT (final—initial evaluation) was observed in the Intervention Group compared to Control (−3.65 ± 3.61 vs +2.27 ± 7.18, p < 0.001) (Table 3).
Parallel to these improvements in functional and static evaluation, a reduction in the number of falls/patient (final-initial evaluation) was observed in the Intervention Group compared to Control (−0.77 ± 1.76 vs +0.03 ± 0.98, p = 0.018) (Table 3).
Discussion
Few studies have been developed regarding balance training in patients with osteoporosis. The present longitudinal prospective study demonstrated that a program of balance training realized over a period of 12 months was effective in improving the functional and static balance, mobility and diminishing the number of falls in elderly women with osteoporosis.
In the present work, improvement in relation to functional balance was demonstrated by an increase in the BBS score in the final evaluation in the group submitted to the balance training program (Intervention Group). Similar results were shown in a study by Melzer et al. [32], where the patients who participated in the balance training obtained 64% improvement in 3 months. The authors showed that the group who underwent the balance training demonstrated better performance than the group submitted to muscular strength training.
Programs that emphasize balance training are more effective at improving balance than those that consist primarily of aerobic, muscular strength or flexibility exercises [33].
Although it is a complex issue to evaluate the effectiveness of different types of exercises [34], balance training has shown beneficial results, with diminished risks of falls [35].
Another positive result of the present study was the improvement in the two difficult conditions of the Clinical Test of Sensory Interaction for Balance (condition 5: eyes closed, unstable surface, and condition 6: eyes open, visual conflict, unstable surface) in almost half the patients. Carter et al. [24] obtained an improvement in static balance using muscular strength training rather than balance training, though only in 6.3% of the patients. Thus, the present findings suggest that balance training leads to more evident positive results in static balance than does muscular strength training.
In relation to functional mobility, improvement was demonstrated by a reduction in the TUGT time in the patients submitted to intervention. These results are relevant, since research shows that compromised mobility increases the risk of dependency three- to five-fold, in activities of daily life [36]. This is not surprising, considering that mobility is an important component of daily life activities, for example: going to the shopping mall, to the supermarket, to the doctor or the cinema. Increased dependency could lead to institutionalization and diminished quality of life. Good balance is considered fundamental for improving mobility and preventing falls [36].
In parallel to the improvement in functional evaluation, an important reduction in the frequency of falls was observed. Although the effect of exercise in the prevention of falls in the elderly is yet to be proved, some studies show that physical activity reduces the risk by 40% [37]. On the other hand, in a consensus on prevention of falls in the elderly, the only exercises recommended to prevent falls are those which specifically target balance training [38].
Many studies that show improvement in balance and mobility present similar characteristics in their exercises. This suggests that the content and intensity of the exercise program could be more important than other intervention variables [39]. Moreover, differences in the administration of the exercise program, the professional experience of those who apply the exercises, the location in which the sessions are held and whether the exercise is conducted in group or realized individually, are all fundamental parameters which influence the success of the exercise program[39].
An important factor for the success of the exercise program is adherence. Contrary to the study by Forcan et al. [40], who stated that adherence to exercise in the elderly is weak, in the present study good adherence was observed, with more than half the patients present at all sessions. The current findings are similar to other studies which showed adherence up to 97.5% [17].
The success in adherence in the present study is probably due to the location where the exercises were performed (a pleasant, natural environment), to social interaction, and to the supervision of a physiotherapist. A secure environment, session supervision and the opportunity for social interaction reduce the feeling of isolation. A social support system is considered important in group activities, and helps sustain adherence and the effectiveness of the weekly exercise sessions and also the adherence to home-based exercises [35].
Similarly to our study, Robitaille et al. showed that an exercise program performed in groups improves the balance of the elderly in the community [41].
Another relevant factor was the use of a manual of instructions and illustrations for the realization of home-based exercises, which contributed to the continuity and adherence of the exercises performed. Each exercise was appropriately prescribed and illustrated by a physiotherapist, giving the patient adequate support. Descriptive and illustrated pamphlets have been used in some studies, complemented by a home-based exercise program, with positive results [3, 42].
The present positive results could also be related to the state of health of these patients, which was good in our patients. Buchner et al. [43] showed that exercise can have beneficial effects on health and on the risk of falls in certain subgroups of the elderly.
The physical and psychological benefits of the regularity of the sessions and the environment should never be ignored. Stimulating strategies and demonstrating ability in the transference from one exercise to another, consequently, maintaining enthusiasm during the exercises, can be practiced safely in groups [35].
Although our study did not use laboratory equipment [44] to substantiate the results obtained, we showed by reliable and reproducible scales and tests [26, 28, 29] that balance training performed once a week, supervised by a physiotherapist, and complemented by home-based exercises, is very effective in the improvement of functional and static balance and mobility, and in the reduction of falls in elderly women with osteoporosis.

balance
falls
osteoporosis
mobility
exercise program


Voice quality after endoscopic laser surgery and radiotherapy for early glottic cancer: objective measurements emphasizing the Voice Handicap Index

We analyzed the functional outcome and self-evaluation of the voice of patients with T1 glottic carcinoma treated with endoscopic laser surgery and radiotherapy. We performed an objective voice evaluation, as well as a physical, emotional and functional well being assessment of 19 patients treated with laser surgery and 18 patients treated with radiotherapy. Voice quality is affected both by surgery and radiotherapy. Voice parameters only show differences in the maximum phonation time between both treatments. Results in the Voice Handicap Index show that radiotherapy has less effect on patient voice quality perception. There is a reduced impact on the patient’s perception of voice quality after radiotherapy, despite there being no significant differences in vocal quality between radiotherapy and laser cordectomy.
Introduction
Surgery and radiotherapy offer the same results in the treatment of early glottic carcinoma, with a cure rate of approximately 90% [1].
The introduction of endoscopic CO2 laser surgery in early glottic carcinoma (epidermoid carcinoma confined to the true vocal cords with normal mobility) has added controversy to the standard treatment choice. Laser surgery is quicker, which reduces cost considerably. However many institutions prefer to use radiotherapy in the belief that the voice will be better preserved after treatment [2]. Although the principal objective of oncology treatment is the complete eradication of the illness, normal voice preservation is another important consideration in the treatment choice of early glottic carcinoma. For this reason, post-treatment voice quality is a relevant factor to take into account when evaluating the results.
Quantitative acoustic measurements are more regularly studied. These are obtained from tools that digitize and analyze the voice being investigated and quantify the characteristics that deviate from normality, understanding normality to mean when the voice is uniform in both amplitude and tone periodicity. The addition of noise to the voice signal is also a defect that affects voice quality, and this is the third acoustic characteristic that is studied using spectrography.
Another of the factors to take into account is the auditive perception that is generated in the listener and is evaluated by ways of perceived voice quality, making use of semi-objective scales such as the GRBAS scale, as described by Hirano [3]. It is recommended that these measurements are made by two experts, although the parameters have shown sufficient reliability (inter- and intra-observer reproducibility) when used in a clinical setting [4].
Although all these measurements are important parameters in defining voice quality, they fail to provide information on the patient’s perceived voice quality. For this there is a method available that permits the patient to describe the sensations their voice gives: the Voice Handicap Index [5]. It is a questionnaire that reviews situations grouped into three areas (functional, physiological and emotional) and gives an idea of the subjective impact that a vocal problem produces in a specific individual.
In this study, we present the objective and subjective analysis of voice quality following treatment of an early epidermoid glottic carcinoma. Results from the objective evaluation of the voice, along with the self-evaluation of voice quality quantified using the Voice Handicap Index of a group of patients treated with endoscopic laser surgery are compared with patients treated with radiotherapy.
Materials and methods
Voice analysis and quality of life studies of 19 patients treated with laser surgery and 18 with radiotherapy, suffering from early glottic carcinoma, were evaluated. Lesions were classified according to the American Joint Committee on Cancer [6].
In patients who received radiotherapy as the primary treatment, this was performed using a 6 mv linear accelerator to bilateral ports and one in front, with field sizes ranging from 5 × 5 to 6 × 6 cm. Wedges were used to establish dose homogeneity.
In the case of patients treated with surgery, the treatment consisted of direct laryngoscopy for the complete resection (partial transmuscular cordectomy) of the lesion with CO2 laser. Objective voice analysis and voice quality studies, performed by both patients and examiners by means of an examination protocol including: perceptual analysis of dysphonia, acoustic analysis, aerodynamic efficiency and patient perception, were applied to both groups. In all patients, this protocol was undertaken at least 6 months after completing treatment.
Perceptual analysis of dysphonia (GRBAS)
Perceptual analysis of dysphonia was performed using the GRBAS scale [3]. Two experienced professionals evaluated the recorded voice samples simultaneously, classifying each sample from 0 to 3 (0 = normal, 1 = mild, 2 = moderate, 3 = severe). The severity of hoarseness is quantified under the parameter G (grade), which represents overall voice quality. B (breathiness): audible impression of turbulent air leakage through an insufficient glottic closure, which may include short aphonic moments (unvoiced segments). R (roughness or harshness): audible impression of irregular glottic pulses, abnormal fluctuations in F0, separately perceived acoustic impulses (as in vocal fry), includes diplophonia and register breaks. A (asthenicity): impression of weakness in the spontaneous phonation, hypokinetic or hypofunctional voice. S (strain, vocal tension): auditive impression of excessive force or tension associated with spontaneous phonation.
Acoustic and spectrographic analysis
The acoustic analysis was performed using Doctor Speech V3 software for Windows 95. The acoustic signal was recorded using the Voice Assessment application. The computer used was a Pentium II at 100 MHz and with 16 Mb of RAM and a Sound Blaster 16 sound card. The sampling frequency was 44,100 Hz and a high frequency range microphone was used. The microphone was located 10 cm from the patient’s mouth while they emitted the “e” sound at comfortable intensity and pitch levels in a soundproof chamber. The computer captured 3 s of sound. Once the signal was digitized, the computer calculated the following acoustic parameters: fundamental frequency (F0), jitter or frequency variation (%), shimmer or amplitude variation (%) and NNE or normalized noise energy, which measures the degree of noise produced by turbulent air escaping through the glottis during vocal emission.
Using the same digitized voice sample, a narrow band spectrogram was generated (45 Hz) using the Speech Analysis application. The spectrograms obtained were grouped into four types according to the Yanagihara criteria [7].
Aerodynamic efficiency analysis
This consisted in measuring the maximum phonation time (TMF) for the “a” vocal after instructing the patient to sustain this vocal for the longest time possible at a comfortable pitch and intensity. The patients were asked to repeat the test at least three times and the highest value was retained.
Patient self-perception analysis (Voice Handicap Index)
The patients completed the “Voice Handicap Index” via a self-evaluation form comprising 30 questions covering three domains [5]: functional, physical and emotional, translated from the original work in English by one of the authors. Each question was assigned a score of 0–4 (from least disability to most). In each item, the maximum score was 40 points and we classified them as mild disability (less than 20), moderate (21–30) and severe (more that 30). Adding the three scores together, the maximum possible was 120, and we classified the vocal disability as mild (less than 30), moderate (31–60), severe (61–90) and very severe (91–120).
Statistical analysis
The data obtained was gathered in the statistical database SPSS 12.0. The statistical analysis used was the Student’s t test in order to compare averages and the Chi-squared test for comparing proportions. The statistical differences were considered significant when P was lower than 0.05.
Results
In the patient group treated with CO2 laser, the ages were in the range 44–86 years, with an average age of 64 and a mean follow-up of 30 months (12–48). In the patient group treated with radiotherapy, the ages were in the range 55–81 years, with an average age of 67 and a mean follow-up of 43 months (6–81). All patients in both groups were males.
In both groups, all lesions corresponded to stage I of the TNM classification. In the group of 19 patients treated with surgery, all the lesions were qualified as T1a and in the case of the 18 radiotherapy patients, 13 were classified as T1a and 5 as T1b. No metastases in the neck, or distant, were detected. At the time of the study, all patients were free from illness.
In the group of patients treated with radiotherapy, the nominal total dose was 6,525 cGy, with 225 cGy daily doses for a total of 29 days. Of the PTV volume, 100% received minimum doses of approximately 5,700 cGy, a maximum dose of approximately 6,720 cGy and an average dose of 6,520 cGy.
Aerodynamic efficiency and spectrographic analysis
In Table 1, the maximum phonation times are presented for the “a” vocal along with the quantitative voice analysis of the vocal “e” obtained in the groups treated with laser and with radiotherapy. There was no significant difference between the parameters: fundamental frequency, (F0), jitter, shimmer and NNE on comparing both groups. However, there were significant differences in the maximum phonation time.
Table 1MPT and acoustic analysis of the vocal /e/ after treatment: laser (n = 19) or radiotherapy (n = 18)VariableLaserRadiotherapyMPT11.83 ± 5.288.63 ± 3.23P < 0.05F0 (Hz)173.39 ± 47.41199.04 ± 51.46NSJitter (%)0.44 ± 0.240.72 ± 0.91NSShimmer (%)5.08 ± 4.724.07 ± 4.04NSNNE (dB)−5.82 ± 2.98−5.02 ± 4.44NSMPT maximum phonation time, F0 (Hz) fundamental frequency, NNE normalized noise energy
In spectrographic terms, the 19 voice samples of patients treated with laser were classified as Grade I 4 (21%), Grade II 5 (25%), Grade III 7 (37%) and Grade IV 3 (15%). In the group of patients treated with radiotherapy, the 18 voice samples were classified as Grade I 1 (5%), Grade II 7 (39%), Grade III 5 (28%) and Grade IV 5 (28%). On comparing the spectrograms obtained in both groups, there were no statistically significant differences (P = 0.401).
Perceptual dysphonia analysis (GRBAS)
Table 2 shows the results obtained using GRBAS scale on patients treated with CO2 laser surgery. Mild dysphonia was found in 31.5% of the cases, moderate dysphonia in 37% and severe dysphonias in the remaining 31.5%. Table 3 shows the GRABS results of the patients treated with radiotherapy. Normal voices were observed in 11%, mild dysphonias in 44.4%, moderate dysphonia in 27.8% and severe dysphonia in 16.7% of the cases. On comparing the results obtained in the G domain of the GRABS scale, which corresponds with the degree of dysphonia, no significant difference was found between each group (P = 0.309).
Table 2Results obtained using GRBAS scale on the patients treated with CO2 laser surgery (n = 19)0123TotalG0%31.5% (6)37% (7)31.5 (6)100% (19)R10.5% (2)58% (11)26% (5)5.5% (1)100% (19)A21% (4)42% (8)21% (4)16% (3)100% (19)B84% (16)16% (3)0%0%100% (19)S31.5% (6)31.5% (6)21% (4)16% (3)100% (19)G grade, R roughness, A asthenicity, B breathiness, S strainTable 3GRABS results of patients treated with radiotherapy (n = 18)0123TotalG11.1% (2)44.4% (8)27.8% (5)16.7%(3)100% (18)R55.6% (10)33.3% (6)5.6% (1)5.6% (1)100% (18)A55.6% (10)33.3% (6)11.1% (2)0%100% (18)B44.4% (8)27.8% (5)22.2% (4)5.6% (1)100% (18)S77.8% (14)16.7% (3)5.6% (1)0%100% (18)G grade, R roughness, A asthenicity, B breathiness, S strain
Patients self-perception analysis (Voice Handicap Index)
Table 4 shows the averages obtained from the “Voice Handicap Index” questionnaire in the functional, physical and emotional scales, as well as the scores obtained in both groups. Upon completing the comparison between the two groups, the statistical difference is significant, in favor of the radiotherapy patients in the functional and emotional ratings as well as the global scores. No significant differences were found in the physical scales.
Table 4Averages obtained from the “Voice Handicap Index” questionnaire in the functional, physical and emotional scales, as well as the scores obtained in both groupsLaser RadiotherapyFunctional11.47 (0–32)2.83 (0–17)P < 0.05Physical12.68 (0–31)6.22 (0–18)NSEmotional4.63 (0–20)0.61 (0–5)P < 0.05Global28.79 (0–77)9.67 (0–29)P < 0.05
Discussion
Early glottic carcinoma can be treated using endoscopic surgery, radiotherapy or partial open surgery. Lesions limited to the vocal fold are normally treated with endoscopic surgery or partial open surgery, with local control results of between 80 and 90% [8]. In choosing one treatment or another, one should contemplate the cure rate, larynx preservation rate, post-treatment voice quality, morbidity and treatment cost [9].
In recent years, endoscopic CO2 laser surgery has made headway compared to radiotherapy, based on its good oncology results and reduced morbidity. CO2 laser treatment in early glottic carcinoma has greatly improved voice quality compared to that obtained following cordectomy via laryngofissure. as documented by some authors such as Keilmann et al. [10]. However in other published studies, such as Schindler [11], this improvement is not as evident. One of the advantages of laser surgery is its low cost when compared to radiotherapy, as well as the additional benefit of being able to opt for radiotherapy at a later stage to treat a relapse or second primary malignancy. In many institutions, these tumours are treated with external radiotherapy because of the supposedly better functional and quality of life results obtained compared to patients treated with surgery [12].
Various studies have been published comparing voice quality after both treatments [13–15]. In some studies, voice quality is similar, while other authors maintain that the voice is better after radiotherapy than after laser surgery [16–18]. However, there are fewer published works that include patient opinions with regard to the impact the illness and the treatment has had on their quality of life [19–21].
In our work, no significant differences in fundamental frequency (F0), jitter, shimmer and NNE were found between the two groups. There were, however, significant differences in maximum phonation time, favoring the patients treated with CO2 laser cordectomy. Other authors do not find significant differences in the maximum phonation time between the groups [22–24]. In the study by Tamura et al. [13] the fundamental frequency is higher for patients treated with CO2 laser surgery, which suggests that this surgery has a greater impact on the vocal fold function than radiotherapy. Krengli et al. [18] found statistically significant differences in the vocal acoustic parameters and fundamental frequency, favoring radiotherapy patients.
In terms of the spectrograms obtained in both groups, no statistically significant differences were found. According to our results, 48% of patients treated with laser, and 44% of radiotherapy patients have an aesthetically acceptable voice (type I and II dysphonia).
There were no significant differences found between the two groups on comparing the scores obtained using the G parameter of the GRABS scale, which corresponds to the degree of dysphonia. Other studies such as Loughran et al. [20] failed to find statistically significant differences on comparing GRABS between patients treated with each technique. In our work, there is an elevated percentage of patients treated with CO2 laser (84%) who obtained low scores in the breathy voice domain (B), which shows that apart from individual healing patterns, or treatment-induced secondary glottic defects, post-cordectomy phonetic compensating mechanisms are important in determining new voice quality. According to Sittel et al. [25], glottic phonation voice quality is better than that obtained by the non-glottic phonation. Although one could believe that these patients had benefited from voice therapy, there are contradictory opinions on this [26].
While acoustic, physiological and perceptual measurements are important parameters in assessing vocal function, they do not provide information about the patient’s perception of their own voice quality. Given that voice quality, because of its potential impact on quality of life, can be an important factor in the choice of treatment, it is important to include this information when evaluating results. Not only should dysphonia as a by-product of laryngeal physiology be considered, but also the effects on patient quality of life must be considered as well.
In our study, in order to quantify patient opinion as to the impact caused by treatment, the Voice Handicap Index has been employed. This is an instrument developed to help the professional in deciding the therapy, taking into account the patient’s subjective sensations with respect to their problem. It is a post-treatment global result evaluation method, from the point of view of the patient’s perceived global well being (physical, mental and social). Although it is a subjective evaluation based on the patient’s own perception, it can provide valuable data as to the reasons why patients with similar dysphonia characteristics can have different handicap severity indices [27]. VHI allows investigators to obtain information regarding the patient’s subjective perception as well as providing the physician with important information pre and post treatment. Given that the preservation of adequate phonation is an important consideration in early glottic carcinoma treatment selection and given equal oncologic results between the two treatments, the VHI proves to be a very important tool in the therapeutic decision-making process.
In our study, low scores were obtained for both groups in the three scales, functional, physical and emotional. This could imply reduced impact in the quality of life of patients treated either with laser surgery or radiotherapy. The statistically significant differences are in favor of radiotherapy patients in the functional and emotional scales, as well as the global scores. Although acoustic and perceptual voice analysis in both groups showed no significant differences, the radiotherapy group scored less in the VHI than the surgical group.
Loughran et al. [20] performed a study where they compared the results between both therapies in terms of the Voice Handicap Index as well as two other questionnaires aimed at evaluating the patient’s subjective voice quality (Vocal Performance Questionnaire, VPQ, and Voice Symptom Score, VoiSS). In this work, no significant differences were discovered between radiotherapy and CO2 laser surgery patients in any of the questionnaires, except for the emotional sub-scale inVoiSS.
However, in other studies the average global score in the Voice Handicap Index questionnaire is lower for patients treated with endoscopic laser resection than in patients treated with radiotherapy. Peeters et al. [21] compared the results of both the Voice Handicap Index and a quality of life questionnaire (COOP/WOONCA) following both treatments. In their results, they found statistically significant differences in favor of laser surgery for the VHI and none in the quality of life results. They found higher scores for global VHI results in 40% of patients treated using surgery, and 58% for those treated with radiotherapy. Their conclusions reveal that the treatment for T1 glottic carcinoma frequently generates vocal problems in day-to-day life, influencing negatively certain social activities. This data contradicts the results of our study where VHI scores of both groups were low and the differences found favored the radiotherapy patient group. Recently, Cohen et al. [28] published a meta-analysis in order to classify the quality of life related to voice in patients with TI glottic carcinomas treated with radiotherapy compared with CO2 laser resection. Six studies with a total of 208 patients treated with surgery and 91 with radiotherapy had similar scores for VHI, from which they concluded that both treatments result in comparable vocal handicap levels for patients with T1 glottic carcinomas [28].
In other works such as that by Stoeckli [19], which value long-term results with regard to quality of life (QOL) of early glottic carcinoma patients, no differences were found between the two treatments and they conclude that both therapies provide good results in terms of quality of life. They show in addition, differences in the relative scores for questions such as swallowing, xerostomy and dental problems, favoring patients treated surgically, and show no differences with regard to perceived voice quality. These results could be related to the fact that xerostomy, edema, mucositis and fibrosis increase the sensation of handicap to such an extent that these effects on patient quality of life could be greater than those coming from the dysphonia itself in laser surgery patients.
Few are the patients that have a so-called “normal” voice upon completing radiotherapy, above all taking into account that the larynx of these patients has undergone a surgical procedure in order to get a biopsy, has been intensely exposed to tobacco smoke and belongs generally to aged patients. All these factors negatively affect voice quality [29]. Despite this, the results of our work reveal that radiotherapy treatment for T1 glottic carcinoma generates lower repercussions in the subjective perception of residual dysphonia, as much in functional as in emotional aspects, when compared with CO2 laser surgery. The scores obtained in the VHI were low in the three domains for both groups, from which we can infer a scarce impact on the quality of life for laser surgery and radiotherapy for this type of patient.
Conclusions
Both CO2 laser resection and external radiotherapy as treatment for T1 vocal carcinoma offer similar objective measurement results (acoustic and spectrographic analysis) and subjective measurement results (GRABS scoring). However, in our series, the self-evaluation of the quality of voice, quantified by the VHI, shows a lower impact for radiotherapy patients.

laser
radiotherapy
voice handicap index
glottic carcinoma
cordectomy